Innate lymphoid cells exhibited IL-17-expressing phenotype in active tuberculosis disease

Including 41 healthy subjects (aged 47.49 ± 10.56, female/male = 18/23), 40 active Mtb-infected patients (aged 49.13 ± 19.49, female/male = 10/30; diagnosed by sputum smears or culture method) without anti-Mtb treatment, were recruited from Huadong Hospital Affiliated to Fudan University and the Sixth People’s Hospital of Nantong. The study was approved by Clinical Research Ethics Committee of Huadong Hospital. Clinical information, laboratory test results, blood and written informed consents were collected from each patient.

In the first and second assays (Additional file 1: Fig. S1A), 200 μL whole blood was stimulated with phorbol-12-myristate 13-acetate (PMA, Sigma, MO, USA), ionomycin and Brefeldin A (Biolengend, CA, USA) at 37 °C for 4 h for cell activation. After that, it was incubated protected from light with BV510-viability kit (Zombie Aqua™ Fixable Viability Kit, Biolengend) at room temperature (RT) for 10 min first and then other surface antibody mixture for another 20 min: anti-CD45-APC/Fire 750 (clone HI30, 304061), anti-Lineage Cocktail-FITC (CD3, CD14, CD16, CD19, CD20, CD56, clone UCHT1, HCD14, 3G8, HIB19, 2H7, HCD56, 348801), anti-CD127-BV421 (clone A019D5, 351310), anti-CD294-PerCP/Cy5.5 (clone BM16, 350116), anti-CD117-PE/Cy7 (clone 104D2, 313211). After incubation, it was lysed by lysing buffer (Biolengend) for 15 min at RT and centrifuged 500×g for 5 min. The cell pallet was then fixed with fixation buffer (Biolengend) at RT for 30 min and washed by 1 mL intracellular staining permeabilization wash buffer (Biolengend). The cell suspension in 50 μL permeabilization wash buffer was stained with antibody to cytoplasmic antigen for 30 min again. The first assay intracellular antibody included anti-IL-17A-PE (clone BL168, 512306) and anti-IL-22-APC (clone 2G12A41, 366705); the second assay included anti-IFN-γ-PE (clone 4S.B3, 502508) and anti-IL-5-APC (clone TRFK5, 504305). After washing by 2 mL permeabilization wash buffer, the cell pellet was resuspended in 0.5 mL PBS and was for the further analyses. Helper ILCs were gated as CD45⁺Lineage⁻CD127⁺ cells; ILC1 was CD294⁻CD117⁻ ILCs; ILC3 was CD294⁻CD117⁺ ILCs, and the CD294⁺ ILCs cells were the total ILC2, which consisting of CD117⁺ ILC2 and CD117⁻ ILC2 [17] (Additional file 1: Fig. S1A). The gating of intracellular cytokines was based on the samples without PMA stimulation. (Additional file 2: Fig. S2). For the third assay, the whole blood was incubated with viability kit for 10 min and then surface antibody for another 20 min without stimulation. The surface antibodies included anti-CD45-FITC (clone 2D1, 368508), anti-CD11b-PE/Cy7 (clone CBRM1/5, 301412), anti-CD115-APC (clone 9-4D2-1E4, 347306), anti-CD11c-APC/Cy7 (clone Bu15, 337218), anti-HLA-DR-PerCP/Cy5.5 (clone L243, 307630). After lysis and centrifugation, cell precipitation was resuspended in PBS and for cytometric analysis. In this assay, DCs were defined as CD45⁺HLA-DR⁺CD11c⁺ and macrophages as CD45⁺CD11b⁺CD115⁺ (Additional file 1: Fig. S1B). All antibodies were from Biolengend. Processed samples were analyzed by FCM (FACSAria™ II, BD Biosciences, NJ, USA) and Flow jo™ 10.

Fresh plasma was collected from the blood of 21 healthy subjects, 30 active Mtb-infected patients by centrifuging at 3000r, 10 min. The level of IL-23 in plasma was evaluated by ELISA using human IL-23 ELISA kit (Proteintech, IL, USA) according to manufacturer’s instructions. In brief, 100 μL plasma or standard human IL-23 solution were added to wells and incubated for at 37 °C for 2 h followed by washing with wash buffer for 4 times. Then 100 μL detection antibody was added and incubated at 37 °C for 1 h. After 4 times washing, every well was incubated with 100 μL diluted streptavidin-HRP solution at 37 °C for 40 min and washed by wash buffer for 4 times. Then 100 μL tetramethylbenzidine (TMB) substrate was added and incubated at 37 °C for 20 min in dark, which was stopped by 100 μL stop solution. Then Synergy H1 microplate reader (BioTek, VT, USA) was used to read the plate at 450 nm and 630 nm immediately. The concentration of plasma IL-23 was calculated based on the established standard curve of IL-23.

Statistical analysis and graphs were performed by SPSS 25.0 software (IBM Corporation, NY, USA) and Prism version 7.00 (GraphPad Software, CA, USA). Data were presented as median with interquartile range. Mann–Whitney U test was used for comparisons between two groups. Rank correlation was used to test the correlation between plasma IL-23 and the cell proportion. Wilcoxon matched-pairs signed rank test was used to compare the IFN-γ⁺ cells and IL-17⁺ cells out of ILC1 in TB group. Clinical information including age, BMI, symptom score and hemogram indices of TB group was presented as mean ± SD (standard deviation) in Table1, and was compared by Student’s t test. Sex and first symptom were compared by Chi-square test. Two-sided P < 0.05 was considered significant.

ILCs are an essential cell group reacting quickly to microorganism. We first tested the proportion of ILCs in blood. As shown in Fig. 1a, the median of ILCs in TB group was 0.73%, which was more than that in NC group (0.42%) significantly (P = 0.0019). As shown in the typical contour map of two groups (Fig. 1b), we found that ILC1 was increased in active Mtb-infected individuals (0.55%) compared with that in healthy subjects (0.31%, Fig. 1c, P = 0.0024). Similarly, CD117⁺ ILC2 in TB group was also increased (0.02% vs. 0.01%, Fig. 1e P = 0.0267). CD117⁻ ILC2 and ILC3 were unchanged (Fig. 1d, f). Therefore, circulatory ILCs were upregulated in active TB disease, and the major expansion was in ILC1 and CD117⁺ ILC2 subsets.

IL-17 had been reported to be critical for Mtb control [10, 18]. In addition to derivation from Th17 cells, IL-17 from ILCs is also respectable. IL-22 is another important proinflammatory cytokine that mediates host defense against pathogens. In ILCs, ILC3 was the major producer of IL-17 and IL-22, which were demonstrated to be the critical inducers for protective innate immunity against Mtb in lung [3, 12]. Besides, CD117⁺ ILC2 was found to have ILC3-like characteristics—bearing Th17 cytokines-producing potential [8, 19]. Consequently, we analyzed the intracellular expression of IL-17 and IL-22 in the peripheral blood by FCM. In line with our expectation, the median of IL-17⁺ CD45⁺ lymphocytes in TB group was 3.83%, significantly more than that in NC group of 1.76% (Fig. 2b P = 0.0006), though the level of IL-22⁺CD45⁺ lymphocytes showed no difference (Fig. 2b). Next, we analyzed the proportion of producing IL-17 and IL-22 cells in ILC subsets. It can be seen that ILC1, CD117⁺ ILC2, ILC3 in TB group all had increased production of IL-17, presenting as the relative frequencies of IL-17⁺ cells (8.79%, 19.01%, 15.15%, respectively) were several times as much as those in NC group (3.87%, 4.57%, 4.55%, Fig. 2c, e, f, P < 0.0001). CD117⁻ ILC2 had no difference in producing IL-17 between two groups (Fig. 2d). And all ILC subsets had unchanged expression of IL-22 (Fig. 2c–f). These results together implied an enhanced ability to express IL-17 of circulatory ILCs in active TB disease.

Based on the concept that ILC1 is akin to Th1 and ILC2 to Th2, we also tested the function of ILCs to generate Th1 cytokine IFN-γ and Th2 cytokine IL-5. In the whole, the relative frequency of IFN-γ⁺ lymphocytes were 9.41% in TB group, twice more than that in NC group of 3.25% (Fig. 3b, P < 0.0001). In ILCs compartment, the production of IFN-γ in ILC1 was dramatically induced in Mtb infection, from 0.56 to 4.37% (Fig. 3c, P = 0.0002). CD117⁻ ILC2 had undetectable IFN-γ production (data was not shown); CD117⁺ ILC2 and ILC3 had unchanged expression of IFN-γ (Additional file 3: Fig. S3A–C). As for IL-5, there was no significant difference between two groups in total IL-5⁺ lymphocytes (Fig. 3b). Additionally, ILC1, CD117⁻ ILC2, CD117⁺ ILC2 all had similar expression of IL-5 between two groups (Fig. 3c, Additional file 3: Fig. S3A, D, E). ILC3 had almost undetected expression of IL-5 (data was not shown). Due to that ILC1 had both enhanced expression of IL-17 and IFN-γ, we analyzed which cytokine dominated in ILC1. As shown in Fig. 3d, IL-17⁺ ILC1 (8.79%) was more than IFN-γ⁺ ILC1 (4.37%) in TB group (P < 0.0001), which implied a bias of ILC1 into IL-17-expressing phenotype in active TB disease.

Previous studies had reported that the production of IL-17 and IL-22 was dependent on stimulation with IL-23 [9, 20]. Consequently, we tested the level of IL-23 in plasma in two groups (21 healthy subjects, 30 active TB patients). The median of plasma IL-23 in two groups was 5.564 pg/mL, 7.551 pg/mL, respectively. Though no statistical meaning was detected, TB group had an increasing tendency of plasma IL-23 than NC group (Fig. 4a, P = 0.0557). Then we analyzed the relationship between plasma IL-23 and features of ILCs in TB group by using rank correlation. Firstly, we analyzed whether IL-23 was associated with number of CD117⁺ ILCs in active TB disease. We found the number of CD117⁺ ILC2 and ILC3 had no correlation to plasma IL-23 (Additional file 4: Fig. S4A, B). However, the ability to produce IL-17 of CD117⁺ ILC2 (Fig. 4b, r = 0.4656, P = 0.0095), ILC3 (Fig. 4c, r = 0.4767, P = 0.0077) were related to plasma IL-23. Surprisingly, the proportion of IL-17⁺ ILC1 to total ILC1 was also correlated to plasma IL-23 (Fig. 4d, r = 0.3937, P = 0.0314). Consequently, it could be surmised that the IL-23 may be the potential inducer for production of IL-17 from ILCs in active TB disease.

Antigen-presenting cells (APCs), including macrophages and DCs, were the major sources of IL-23 when they were activated by pathogens. It has been well-established that tissue migratory macrophages and dendritic cells in response to stimulation are derived from monocytes in circulation [21]. During inflammation, circulating monocytes are predestined to become activated macrophages and DCs with some upregulated surface markers [21]. Therefore, we tested whether circulatory monocytes were stirred in active Mtb infection. Based on previous studies [21‐23], here we defined HLA-DR⁺CD11c⁺ cells as DCs, CD115⁺CD11b⁺ cells as macrophages (Additional file 1: Fig. S1B) in the third assay. Compared with healthy subjects, patients with active Mtb infection showed higher level of DCs (9.35% vs. 6.49%, Fig. 5a, P < 0.0001) but similar level of macrophages (Fig. 5b). It suggested that DCs in vivo was triggered by Mtb for the initial defense.

As mentioned before, ILCs in TB patients exhibited IL-17-expressing phenotypes compared with NC group, we again compared the proportion of IL-17⁺ ILCs out of total ILCs between two groups. As shown in Fig. 6a, ILCs in TB group had evident upregulated expression of IL-17 (9.81% vs. 4.00%, P < 0.0001). We then analyzed whether this phenotype was associated with clinical symptoms and laboratory measurements. According to the median of IL-17⁺ ILCs, we divided TB patients into two groups, and the clinical information was listed in Table1. Clinical symptoms including cough, sputum, fever, purulent sputum, bloodshot sputum, chest pain and loss of weight were scored 1 unanimously and then added up to a total symptom score. Patients with higher frequency of IL-17⁺ ILCs tend to have more white blood cells (P = 0.1000) and neutrophils (P = 0.0952) but less eosinophils (P = 0.0047). Moreover, this group had obvious poor clinical condition with lower red blood cells (P = 0.0158), hemoglobin level (P = 0.0278) but higher platelet level (P = 0.0373). Though the proportion of patients reporting clinical symptoms had no significant difference between two groups (Fig. 6b, P = 0.3780), patients with higher IL-17⁺ ILCs still tended to get higher symptom scores (Table1, P = 0.1314). Because of the similar lung mass or nodules in pulmonary images between lung TB and lung cancer, some patients were examined plasma cancerous indices such alpha-fetoprotein (AFP), CA199, carcino embryonic antigen (CEA), and inflammatory indices such as lactate dehydrogenase (LDH), C-reactive protein (CRP), adenosine deaminase (ADA) for differential diagnosis. We found that patients with higher IL-17⁺ ILCs tended to harbor higher CRP but lower CA199 and AFP. The specific marker for Mtb infection ADA was not different between two groups (Fig. 6c). These results indicated that IL-17⁺ ILCs was associated with inflammatory status and poor condition of active TB patients.