Up-regulated SNHG16, TLR4 and TRAF6 expression with increased apoptosis, autophagy and NETs formation in SLE-AH lung tissues

Representative H&E-stained lung tissues from a PTX control patient and 3 AH patients with underlying SLE, IgAV or AAV were shown in Fig. 1a. The AH lungs were examined for the expression of apoptosis, autophagy and NETs formation. In Figs. 1b, 2a, higher numbers of TUNEL-positive cells were found in lung tissues from SLE-AH patients than from PTX controls (40.3 ± 6.1 versus 1.4 ± 1.1, p = 0.036). In Fig. 1c, 2a, colocalized expression of CitH3 with DNAs, in favor of NETosis, was identified in the SLE-AH lungs but not in the PTX (18.0 ± 6.0 versus 0 ± 0, p = 0.018), or IgAV-AH lungs. In particular, binding of ANCA to myeloperoxidase expressed on the membrane of pulmonary neutrophils could be responsible for distinct NETosis in AAV-associated AH lung tissues (Figs. 1c, 2a) [36]. In Fig. 1d, 2a, cytoplasmic LC3-positive cells, suggesting autophagy formation, were identified in SLE-AH but not in PTX (Fig. 2a, 7.7 ± 3.1 versus 0 ± 0, p = 0.018), IgAV-AH or AAV-AH lung tissues.

Fig. 1

Increased apoptosis, autophagy and NETs formation in SLE-associated AH lung tissues. a From top to low, representative histopathology from a PTX control and AH patients from SLE, IgAV and AAV. In PTX, normal alveoli. In SLE, AH with blood in alveoli. In IgAV, AH with interstitial lymphoplasmatic cells and fibrosis. In AAV, AH with fibroblastic plugs in alveoli and alveolar ducts. H&E staining, scale bar = 100 µm and magnification ×100. b From top to low, representative TUNEL IF staining (green) from a PTX control and AH patients from SLE, IgAV and AAV. Cell nuclei counterstained with DAPI (blue). Scale bar = 25 µm, magnification ×400. c From top to low, representative CitH3 IF staining (green) from a PTX control and AH patients from SLE, IgAV and AAV. Cell nuclei counterstained with Hoechst 33258 (blue). Scale bar = 12.5 µm, magnification ×800. d From top to low, representative LC3 IF staining (green) from a PTX control and AH patients from SLE, IgAV and AAV. Arrows pointing cells with positive cytoplasmic LC3 staining. Cell nuclei counterstained with Hoechst 33258 (blue). Scale bar = 10 µm, magnification ×1000 for SLE. Scale bar = 12.5 µm, magnification ×800 for PTX, IgAV and AAV

Fig. 2

Up-regulated cell death processes and pulmonary expression of SNHG16, TLR4 and TRAF6 in SLE-associated AH lung tissues. a Quantification of cell numbers with positive TUNEL, colocalized CitH3 and DNAs, and cytoplasmic LC3 in lung tissues from PTX controls and AH patients from SLE, IgAV and AAV. Expression levels of b SNHG16, c TLR4, d TRAF6, e miR-146a and f NEAT1 in lung tissues from PTX controls and AH patients from SLE, IgAV and AAV. Expression levels of g p53, h Bax, i HGMB1, j PAD4, k LC3, l Beclin-1, m mTOR, n p62, o IL-6, p IL-8, q IFN-α and r MX-1 in lung tissues from PTX controls and SLE-AH patients. Lung sample numbers, n = 5 for PTX, n = 3 for SLE, n = 1 for IgAV, n = 1 for AAV. Values are mean ± SD. Horizontal lines are mean values. *p < 0.05

There were up-regulated SNHG16, TLR4 and TRAF6 expression (Fig. 2b–d, p  = 0.036), down-regulated miR-146a expression (Fig. 2e, p = 0.036), and no differences in NEAT1 expression (Fig. 2f) in SLE-AH lung tissues. Up-regulated levels of cell death-related markers p53, Bax, HMGB1, PAD4, LC3 and Beclin-1 were found in lung tissues from SLE-AH patients (Fig. 2g–l, p = 0.036). For other autophagy-related markers, there was down-regulated expression of mTOR (Fig. 2m, p = 0.036), an autophagy initiation inhibitor by reducing the activity of autophagy regulatory complexes [23], whereas no differences were found in p62 levels (Fig. 2n). In addition, there were increased levels of cytokines IL-6, IL-8 and IFN-α as well as IFN-inducible gene MX-1 in SLE-AH lung tissues (Fig. 2o–r, p = 0.036).

Collectively, these findings suggested up-regulated SNHG16 expression with a synchronously increased TLR4 and TRAF6 levels to enhance apoptosis, autophagy and NETs formation in the SLE-associated AH lungs.

Up-regulated SNHG16, TLR4 and TRAF6 levels in PBMCs from SLE-AH patients

PBMCs from SLE patients were examined for the expression of SNHG16. Higher levels were found in SLE patients than in HC (Fig. 3a, 352.1 ± 682.4% versus 100.0 ± 174.6%, p = 0.006). A positive correlation was found between SNHG16 levels and activity scores (Fig. 3b, r = 0.409, p = 0.001). For SNHG16 expression in different patient groups and HC, SLE-AH had higher levels than AH from other autoimmune diseases, LN, Nil or HC (Fig. 3c, p = 0.001 for other AH, p = 0.038 for LN, p < 0.001 for Nil or HC). A negative correlation was found between miR-146a and SNHG16 levels (Fig. 3d, r = − 0.334, p = 0.008),

Fig. 3

Up-regulated SNHG16, TLR4 and TRAF6 expression in PBMCs from SLE-AH patients. a SNHG16, e TLR4 and i TRAF6 levels in PBMCs from HC and SLE patients. A positive correlation between PBMC b SNHG16, f TLR4 and j TRAF6 levels and SLEDAI-2K activity scores. c SNHG16, g TLR4 and k TRAF6 levels in PBMCs from HC, Nil, LN, SLE-AH and other AH. A positive correlation between SNHG16 and d miR-146a, h TLR4 or l TRAF6 levels in PBMCs from SLE patients. Values are mean ± SD. Horizontal lines are mean values. Patient numbers, n = 62 for SLE, n = 7 for Nil, LN, SLE-AH, n = 6 for other AH. *p < 0.05, **p < 0.01, ***p < 0.001

TLR4 levels in PBMCs was higher in SLE patients than in HC (Fig. 3e, 211.5 ± 194.8% versus 100.0 ± 61.3%, p = 0.001). A positive correlation was found between TLR4 levels and activity scores (Fig. 3f, r = 0.304, p = 0.017). For TLR4 expression, SLE-AH had higher levels than other AH, LN, Nil or HC (Fig. 3g, p = 0.001 for other AH, p = 0.038 for LN, p = 0.002 for Nil, p < 0.001 for HC). A positive correlation was found between TLR4 and SNHG16 levels (Fig. 3h, r = 0.384, p = 0.002), but not between TLR4 and NEAT1 levels (Additional file 1: Fig. S4a).

The expression of TRAF6, a TLR4 downstream signaling molecule, was higher in SLE patients than in HC (Fig. 3i, 151.1 ± 76.3% versus 100.0 ± 45.3%, p = 0.002). A positive correlation was found between TRAF6 levels and activity scores (Fig. 3j, r = 0.365, p = 0.004). For TRAF6 expression, SLE-AH had higher levels than other AH, LN, Nil or HC (Fig. 3k, p = 0.001 for other AH, p = 0.026 for LN, p = 0.001 for Nil, p < 0.001 for HC). A positive correlation was found between TRAF6 and SNHG16 levels (Fig. 3l, r = 0.272, p = 0.033).

Furthermore, there were significant differences by using multivariable analyses adjusted for age and sex for the comparison of SNHG16, TLR4 or TRAF6 levels between SLE and HC, while a significant positive correlation was found by using multivariable analyses adjusted for age, sex and medications for the comparison of SNHG16, TLR4 or TRAF6 levels with activity scores (Additional file 2: Table S2).

In addition, PBMC subpopulations were examined for the expression of SNHG16 in sorted CD3-positive T cells, CD19-positive B cells and CD14-positive monocytes from each healthy individual (Additional file 1: Fig. S2b). In comparison with the average expression levels of SNHG16 in neutrophils from 3 healthy individuals, there were no differences in T cells, whereas lower levels were found in monocytes and B cells (For neutrophils, 100.0 ± 2.0%, for T cells, 85.1 ± 14.0%, for monocytes, 36.3.0 ± 16.3%, p = 0.002, for B cells, 30.1 ± 6.4%, p = 0.002). We also examined the actual cell number of PB neutrophils, T cells, monocytes and B cells in each healthy individual (Additional file 1: Fig. S2b). There were higher circulating numbers of neutrophils, followed by T cells, monocytes and B cells in each healthy individual.

PBMCs from SLE patients were also examined for the expression of miR-146a. Lower miR-146a levels were found in SLE patients than in HC (Additional file 1: Fig. S3a, p < 0.001). A negative correlation was found between miR-146a levels and activity scores (Additional file 1: Fig. S3b, r = − 0.366, p = 0.003). For PBMC miR-146a expression, SLE-AH had lower levels than other AH, LN, Nil or HC (Additional file 1: Fig. S3c, p = 0.001 for other AH, p = 0.026 for LN, p < 0.001 for Nil or HC). Furthermore, a negative correlation was found between miR-146a and TLR4 (Additional file 1: Fig. S3d, r = − 0.290, p = 0.022), TRAF6 (Additional file 1: Fig. S3e, r = − 0.404, p = 0.001), or NEAT1 levels (Additional file 1: Fig. S3f, r = − 0.256, p = 0.045).

We further examined the expression of NEAT1, another lncRNA involved in the SLE activity and also a ceRNA targeting miR-146a [30], in PBMCs from SLE patients. Higher levels were found in SLE patients than in HC (Additional file 1: Fig. S4b, 253.8 ± 428.8% versus 100.0 ± 96.9%, p = 0.011). A positive correlation was found between NEAT1 levels and activity scores (Additional file 1: Fig. S4c, r = 0.306, p = 0.016). For NEAT1 expression in different patient groups and HC, AH had higher levels than HC (Additional file 1: Fig. S4d, p = 0.038), whereas no differences were found between SLE-AH and other AH, LN or Nil. Although both lncRNAs had up-regulated expression in SLE patients and acted as ceRNAs targeting miR-146a, SNHG16 rather than NEAT1 appeared to be involved in the pathogenesis of AH manifestation.

Taken together, these results implicated that up-regulated SNHG16 levels with a concurrent increase in the expression of TLR4 and TRAF6 in PBMCs can participate in SLE activity, resulting in the AH manifestation.

Increased cell death processes in PB leukocytes from SLE-AH patients

In SLE, accelerated apoptosis has been identified in PB lymphocytes and monocytes [3], while increased p53 levels and cell apoptosis in circulating lymphocytes are correlated with the disease activity [35]. The excessive activation of autophagy leads to autophagic cell death, and the expression of autophagy flux-related molecules LC3 and Beclin-1 has been found to be increased in PBMCs from SLE patients [3, 37]. There are overactivated neutrophils with increased NETs formation in PMA-stimulated PBNs from SLE patients [3, 24].

SLE patients had higher p53 levels (Additional file 1: Fig. S5a) and apoptotic cell ratios (Additional file 1: Fig. S5b) in PBMCs than those from HC (For p53, p = 0.004; for apoptotic cell, p = 0.012). Furthermore, SLE-AH had higher p53 levels (Additional file 1: Fig. S5a) and apoptotic cell ratios (Additional file 1: Fig. S5b) than those from LN, Nil or HC (For p53, p = 0.038 for LN, p = 0.004 for Nil, p < 0.001 for HC; for apoptotic cell, p = 0.036 for LN, Nil or HC).

There were higher LC3 and Beclin-1 levels and lower mTOR levels in PBMCs from SLE patients than those from HC (Fig. 4a, for LC3, p = 0.002, Fig. 4d, for Beclin-1, p = 0.004, Fig. 4g, for mTOR, p = 0.047), while LC3 and Beclin-1 levels were positively correlated with activity scores (Fig. 4b, for LC3, r = 0.359, p = 0.004; Fig. 4e, for Beclin-1, r = 0.399, p = 0.001). No differences in p62 levels were found between SLE patients and HC (Fig. 4j). For LC3, AH had higher levels than Nil or HC (Fig. 4b, p < 0.001 for Nil or HC). For Beclin-1, AH had higher levels than LN, Nil or HC (Fig. 4c, p = 0.038 for LN, p < 0.001 for Nil or HC). For mTOR, AH only had lower levels than HC (Fig. 4i, p = 0.018). In LPS-stimulated PBMs, AH only had higher LC3 levels than HC but not other patient groups (Fig. 4k, p = 0.036), whereas AH had higher Beclin-1 levels than LN, Nil or HC (Fig. 4l, p = 0.036 for LN, Nil or HC). In addition, we performed immunoblotting assay to examine the protein expression of LC3, Beclin-1 and mTOR in PBMCs from SLE patients and HC. SLE patients had higher LC3-II and Beclin-1 but lower mTOR expression than those from HC (Additional file 1: Fig. S6a, representative immunoblot assay for Beclin-1, LC and mTOR in PBMCs from SLE patients and HC; Fig. S6b, p = 0.019 for LC-II, p = 0.030 for Becline-1, p = 0.043 for mTOR).

Fig. 4

Increased autophagy formation in PBMCs from SLE-AH patients. a LC3, d Beclin-1, g mTOR and j p62 levels in PBMCs from SLE patients and HC. A positive correlation between b LC3 or e Beclin-1 levels in PBMCs from SLE patients and SLEDAI-2K activity scores. No correlation between h mTOR levels in PBMCs from SLE patients and SLEDAI-2K activity scores. c LC3, f Beclin-1 and i mTOR levels in PBMCs from HC, Nil, LN and AH patients. k LC3 and l Beclin-1 levels in LPS-stimulated PBMs from HC, Nil, LN and AH patients. Values are mean ± SD. Horizontal lines are mean values. Patient numbers, PBMCs, n = 62 for SLE, n = 7 for Nil, LN, AH. Patient numbers, PBMs, n = 5 for Nil, LN, n = 3 for AH. *p < 0.05, **p < 0.01, ***p < 0.001

Up-regulated expression of SNHG16, TLR4 and TRAF6, but not NEAT1, was identified in PBNs from SLE patients in comparison with those from HC (Fig. 5a, d, g, Additional file 1: S4e, for SNHG16, p = 0.045, for TLR4, p = 0.002, for TRAF6, p = 0.026). A positive correlation was found between activity scores and SNHG16 (Fig. 5c), TLR4 (Fig. 5f) or TRAF6 levels (Fig. 5i), but not NEAT1 levels in PBNs (Additional file 1: Fig. S4f) (For SNHG16, r = 0.566, p = 0.028; for TLR4, r = 0.588, p = 0.021; for TRAF6, r = 0.617, p = 0.014). A positive correlation was found between SNHG16 and TLR4 levels (Fig. 5j, r = 0.518, p = 0.048), or TRAF6 levels (Fig. 5k, r = 0.558, p = 0.031). For SNHG16, TLR4 or TRAF6 expression in PBNs, AH had higher levels than LN, Nil or HC (Fig. 5b, 5e, for SNHG16 or TLR4, p = 0.032 for LN, p = 0.016 for Nil or HC; Fig. 5h, for TRAF6, p = 0.016 for LN, Nil or HC). For NEAT1 PBN expression, AH only had higher levels than HC (Additional file 1: Fig. S4g, p = 0.032). In addition, there was lower miR-146a expression in PBNs from SLE patients than those from HC (Additional file 1: Fig. S3g, p = 0.012), while a negative correlation was found between PBN miR-146a and activity scores (Additional file 1: Fig. S3h, r = − 0.539, p = 0.038). AH had lower PBN miR-146a levels than LN, Nil or HC (Additional file 1: Fig. S3i, p = 0.032 for LN, p = 0.016 for Nil or HC).

Fig. 5

Up-regulated SNHG16, TLR4 and TRAF6 expression and increased NETs formation in PBNs from SLE-AH patients. a SNHG16, d TLR4 and g TRAF6 levels in PBNs from SLE patients and HC. b SNHG16, e TLR4 and h TRAF6 levels in PBNs from HC, Nil, LN and AH patients. A positive correlation between c SNHG16, f TLR4 and i TRAF6 levels in PBNs from SLE patients and SLEDAI-2K activity scores. A positive correlation between SNHG16 and j TLR4 or k TRAF6 levels in PBNs from SLE patients. PBNs from HC, Nil, LN and AH patients stimulated with LPS to detect DNAs morphology and measure CitH3/PAD4 levels. l, Left, representative photographs from a HC and an AH patient. Scale bar = 50 µm, magnification ×200. Right, quantification of NETs formation in HC, Nil, LN and AH patients. m Quantification of CitH3 (left) and PAD4 (right) levels in HC, Nil, LN and AH patients. Values are mean ± SD. Horizontal lines are mean values. Patient numbers, n = 15 for SLE, n = 5 for Nil, n = 5 for LN, n = 4 for AH. *p < 0.05, **p < 0.01

In addition, there were significant differences by using multivariable analyses adjusted for age and sex for the comparison of SNHG16, TLR4 or TRAF6 levels between SLE and HC, while a significant positive correlation was found by using multivariable analyses adjusted for age, sex and medications for the comparison of SNHG16, TLR4 or TRAF6 levels with activity scores (Additional file 2: Table S3).

Furthermore, purified PBNs were stimulated with LPS to induce NETosis by observing DNA morphology with NETs formation and measuring CitH3 and PAD4 levels. For spread NETs formation, AH had higher percentages than LN, Nil or HC (Fig. 5l, p = 0.016 for LN, Nil or HC). For CitH3 or PAD4 production, AH had higher levels than LN, Nil or HC (Fig. 5m, for CitH3, p = 0.032 for LN, p = 0.016 for Nil or HC; for PAD4, p = 0.016 for LN, Nil or HC).

Altogether, increased cell death processes including apoptosis, autophagy and NETosis were found in PB leukocytes from SLE patients, particularly in those with the AH manifestation.

Increased Dox-induced apoptosis in SNHG16-overexpressed and reduced apoptosis in SNHG16-silenced MLE-12 cells

Since SLE-AH patients had upregulated SNHG16 expression, increased pro-inflammatory cytokine levels and increased apoptosis formation in their lung tissues, we further investigated whether, in alveolar cells, the presence of pro-inflammatory cytokine could regulate the expression of SNHG16, and the apoptotic status could be altered by overexpressing or silencing the expression of SNHG16. SNHG16 levels in the cultures of MLE-12 cells were upregulated in the presence of exogenous IL-6 with a dose-dependent manner (Additional file 1: Fig. S7a, for 31.3 ng/mL, p = 0.034, for 62.5 ng/mL, p = 0.032, for 125 ng/mL, p = 0.007). Furthermore, in Dox-stimulated MLE-12 cells, there were increased TUNEL-positive apoptotic cell percentages and a dose-dependent increase in apoptotic cell ratios (Additional file 1: Fig. S7b), concentrations of HMGB1, a damage-associated molecular pattern (DAMP) molecule released by apoptotic cells [24] (Additional file 1: Fig. S7c), and levels of SNHG16, TRAF6, p53 and Bax (Additional file 1: Fig. S7d, S7e). In addition, we examined the expression of miR-17 and miR-146a, ceRNA targets of SNHG16 [38], in SNHG16-overexpressed and -silenced MLE-12 cells (Additional file 1: Fig. S7f). Furthermore, SNHG16-overexpressed cells had up-regulated p53, Bax levels and apoptotic cell ratios (Additional file 1: Fig. S7g, for p53 or Bax, p < 0.001, for apoptotic ratio, p = 0.005), whereas SNHG16-silenced cells had down-regulated p53, Bax levels and apoptotic cell ratios (Additional file 1: Fig. S7h, for p53 or Bax, p < 0.001, for apoptotic ratio, p = 0.006).

As a whole, these results suggested that modulating SNHG16 expression could control the Dox-induced-apoptosis via damaging DNAs to trigger a p53-dependent process in alveolar cells.

Increased LPS-induced autophagy in SNHG16-overexpressed and decreased autophagy in SNHG16-silenced MLE-12 cells

Based on the findings of up-regulated LC3/Beclin-1 levels and increased autophagy formation in PB leukocytes and lung tissues from SLE-AH patients, we further modulated the expression of SNHG16 in alveolar cells to examine its influence on the autophagy formation. In LPS-stimulated MLE-12 cells, by using immunoblot and qRT-PCR analyses, there was a dose-dependent increase in LC3-II and LC3 (Fig. 6a) and in Beclin-1 expression (Fig. 6b). In addition, a dose/time-dependent up-regulated SNHG16 and TRAF6 expression and down-regulated miR-146a levels were identified in such cells (Fig. 6c, 6d). Furthermore, SNHG16-overexpressed cells had increased LC3 and Beclin-1 levels (Fig. 6e, p < 0.001), whereas SNHG16-silenced cells had decreased LC3 and Beclin-1 levels (Fig. 6f, p < 0.001). In addition, SNHG16-overexpressed and SNHG16-silenced cells had up-regulated and down-regulated TLR4 expression, respectively, by qRT-PCR analysis and immunoblot assay (Additional file 1: Fig. S8a, TLR4 mRNA, for overexpressed cells, p = 0.002, for silenced cells, p = 0.001; Fig. S8b, representative TLR4 immunoblot assay). These findings indicated that SNHG16 expression could regulate a TLR4/TRAF6 axis-mediated autophagy formation in alveolar cells.

Fig. 6

Autophagy formation in LPS-stimulated MLE-12 cells regulated by SNHG16. a LC3 and b Beclin-1 levels in LPS-stimulated MLE-12 cells. Left, immunoblot assay. Right, qRT-PCR analysis. MLE-12 cells treated with 1 µM rapamycin (Rapa) as a PC. SNHG16, miR-146a and TRAF6 levels in MLE-12 cells stimulated with c different LPS concentrations for 4 h and with d 50 µg/mL LPS for different times. e SNHG16 levels (left) in SNHG16-overexpressed MLE-12 transfectants. LC3 (middle) and Beclin-1 (right) levels in SNHG16-overexpressed transfectants stimulated with 50 µg/mL LPS for 4 h. f SNHG16 levels (left) in SNHG16-silenced MLE-12 transfectants. LC3 (middle) and Beclin-1 (right) levels in SNHG16-silenced transfectants stimulated with 50 µg/mL LPS for 4 h. Values mean ± SD. Results in a–d were representative of 3 independent experiments, and in e and f were representative of 2 independent experiments with similar findings. *p < 0.05, **p < 0.01, ***p < 0.001

TLR4-mediated NETs formation in mouse neutrophils by LPS or HMGB1 stimulation

In PBNs from SLE-AH patients, there was up-regulated SNHG16 expression, while LPS stimulation could enhance NETosis as shown by increased spread NETs formation and CitH3/PAD4 production. On day 4, 9 and 14 after the pristane induction, neutrophils were isolated 24 h later from thioglycolate-injected mice. There was up-regulated expression of SNHG16, TLR4 and TRAF6 as well as down-regulated miR-146a levels since day 4 (Additional file 1: Fig. S9a, on day 4, for SNHG16, p < 0.001, for TLR4, p = 0.002, for TRAF6, p = 0.034, for miR-146a, p = 0.004), but no differences in the NEAT1 expression (Additional file 1: Fig. S4h).

Neutrophils from naïve mice were stimulated with IL-6, LPS or HMGB1 in the cell culture. SNHG16 levels were upregulated under the stimulation of IL-6 in a dose-dependent manner (Additional file 1: Fig. S9b, for 62.5 ng/mL, p = 0.043, for 125 ng/mL, p = 0.018, for 250 ng/mL, p = 0.019). There were increased diffused/spread NETs morphology and CitH3 production, favoring NETs formation, with up-regulated SNHG16 and TRAF6 as well as down-regulated miR-146a expression in HMGB1-stimulated neutrophils in a dose-dependent manner (Additional file 1: Fig. S9c, HMGB1 300 ng/mL, for morphology, p < 0.001, for CitH3, p = 0.035, for SNHG16, p = 0.040, for TRAF6, p = 0.025, for miR-146a, p = 0.036; LPS 3 μg/mL, for morphology, p < 0.001, for CitH3, p = 0.005, for SNHG16, p = 0.003, for TRAF6, p = 0.031, for miR-146a, p = 0.006).

Increased LPS-induced NETosis in SNHG16-overexpressed and reduced NETosis in SNHG16-silenced dHL-60 cells

Differentiated HL-60 cells were stimulated with LPS to induce NETosis as shown by increased diffused/spread NETs morphology and CitH3/PAD4 production (Fig. 7a, for morphology, p < 0.001, for CitH3, p = 0.005, for PAD4, p = 0.001). There were a time-dependent up-regulation of SNHG16 and TRAF6 expression as well as down-regulated miR-146a levels (Fig. 7b, at 1 h, for SNHG16, p = 0.010, for TRAF6, p = 0.042, for miR-146a, p = 0.001). SNHG16-overexpressed dHL-60 cells had higher diffused/spread NETs percentages and CitH3/PAD4 levels (Fig. 7c, for morphology, p = 0.022, for CitH3, p = 0.011, for PAD4, p = 0.016), whereas SNHG16-silenced dHL-60 cells had lower diffused/spread NETs percentages and CitH3/PAD4 levels (Fig. 7d, for morphology, p = 0.024, for CitH3, p = 0.041, for PAD4, p = 0.047).

Fig. 7

NETs formation in LPS-stimulated dHL-60 cells regulated by SNHG16. a LPS-stimulated dHL-60 cells to detect DNAs morphology and measure CitH3/PAD4 levels. Left, representative photographs from mock and LPS stimulation. Scale bar = 60 µm, magnification ×200. Middle left, quantification of diffused/spread NETs morphology percentage. Middle right, CitH3 levels. Right, PAD4 levels. b SNHG16, TRAF6 and miR-146a levels in dHL-60 cells stimulated with 500 ng/mL LPS for different times. c Left, SNHG16 levels in SNHG16-overexpressed HL-60 transfectants. Middle left diffused/spread NETs morphology quantification. Middle right, CitH3 levels. Right, PAD4 levels in SNHG16-overexpressed dHL-60 transfectants stimulated with 50 µg/mL LPS for 4 h. d Left, SNHG16 levels in SNHG16-silenced HL-60 transfectants Middle left, diffused/spread NETs morphology quantification. Middle right, CitH3 levels. Right, PAD4 levels in SNHG16-silenced dHL-60 transfectants stimulated with 500 ng/mL LPS for 4 h. Values are mean ± SD. Results in a and b were representative of 3 independent experiments, and in c and d were representative of 2 independent experiments with similar findings. *p < 0.05, **p < 0.01, ***p < 0.001

SNHG16-overexpressed and SNHG16-silenced HL-60 cells had up-regulated and down-regulated TLR4 expression, respectively (Additional file 1: Fig. S10a, TLR4 mRNA, for overexpressed cells, p = 0.016, for silenced cells, p = 0.001; Fig. S10b, representative TLR4 immunoblot assay). In addition, expression of miR-17 and miR-146a, ceRNA targets of SNHG16 [38], was shown in SNHG16-overexpressed and SNHG16-silenced HL-60 cells (Additional file 1: Fig. S10c). Furthermore, miR-146a expression in SNHG16-silenced HL-60 cells were silenced to elucidate the putative mechanism that up-regulated miR-146a expression is involved in decreased NETosis in LPS-stimulated SNHG16-silenced dHL-60 cells. Sorted CasRX-SNHG16-transfected HL-60 cells were transfected with sh-miR-146a or sh-luciferase to create stable transfectants. After stimulating DMSO-induced dHL-60 cells with LPS, there were higher NETs percentages and CitH3/PAD4 levels in sh-miR-146a-transfected than sh-luciferase-transfected cells (Additional file 1: Fig. S10d, for NETs morphology, p = 0.043, for CitH3, p = 0.030, for PAD4, p = 0.029), implicating that miR-146a expression participates in the regulation of TLR4-mediated NETs formation by SNHG16.

In sum, our experimental data revealed that, in myelocytic cells, a TLR4/TRAF6 axis-mediated NETs formation could be regulated by SNHG16.

Up-regulated pulmonary SNHG16, TLR4 and TRAF6 expression with increased apoptosis, autophagy and NETs formation in a pristane-induced mouse AH model

Figure 8 demonstrates an AH mouse model with complete hemorrhage in 80% of pristane-injected mice and no hemorrhage in PBS-injected controls (Fig. 8a, p < 0.001). Lower RBC numbers, Hb levels and Hct were identified in pristane- than PBS-injected mice (Fig. 8b, p < 0.001). Higher levels of anti-RNP antibody, favoring an IgM isotype, were found in pristane- than PBS-injected mice on day 14 (Fig. 8c, p = 0.038). Up-regulated pulmonary and splenic SNHG16 levels were identified in pristane-injected mice since day 4 (Fig. 8d, day 4, p < 0.001), while upregulated TLR4 and TRAF6 expression and down-regulated miR-146a levels were also found in pristane-injected mice (Fig. 8d, day 4, for pulmonary TLR4, p = 0.006, TRAF6 and miR-146a, p < 0.001, for splenic TLR4, p = 0.043, TRAF6, p = 0.004, miR-146a, p = 0.005). There were no differences between pulmonary and splenic NEAT1 levels in pristane- and PBS-injected mice (Additional file 1: Fig. S4i, S4j). In addition, upregulated expression of IL-6, IL-8, IFN-α and MX-1 was found in pristane-injected mice since day 4 (Fig. 8e, day 4, for IL-6, p = 0.006, IL-8, p < 0.001, IFN-α, p = 0.012, MX-1, p = 0.025).

Fig. 8

Up-regulated SNHG16, TLR4 and TRAF6 expression with increased apoptosis, autophagy and NETs formation in the mouse AH lungs. a Left, representative gross and histopathological photographs in the mouse lungs with no and complete hemorrhage. Right, hemorrhagic frequencies in saline- and pristane-injected mice on day 14. Scale bar = 100 µm, magnification ×100. b RBC numbers, Hb levels and Hct on day 14 in saline- and pristane-injected mice. c Anti-RNP titers on day 14 in serum samples from saline- and pristane-injected mice. d Pulmonary (top) and splenic (low) SNHG16, TLR4, TRAF6 and miR-146a levels on day 0, 4, 9 and 14 from saline- and pristane-injected mice. e IL-6, IL-8, IFN-α and MX-1 pulmonary levels on day 0, 4, 9 and 14 from saline- and pristane-injected mice. f Representative TUNEL IF staining (green) with cell nuclei counterstained with DAPI (blue). Scale bar = 25 µm, magnification ×400. g Representative CitH3 IF staining (green) with cell nuclei counterstained with Hoechst 33258 (blue). Scale bar = 12.5 µm, magnification ×800. h Representative LC3 IF staining (green) with cell nuclei counterstained with Hoechst 33258 (blue). Scale bar = 10 µm, magnification ×1000. Arrows pointing cells with positive cytoplasmic LC3 staining. i Quantification of cell numbers with positive TUNEL, colocalized CitH3/DNAs, and cytoplasmic LC3 in lung tissues. j Immunoblot assay (left) with signal intensity quantitation analysis (right) of pulmonary LC3-II expression from saline- and pristane-injected mice. k Pulmonary levels of p53, Bax, LC3 and Beclin-1 on day 0, 4, 9 and 14 from saline- and pristane-injected mice. Values are mean ± SD. Horizontal lines are mean values. Mouse numbers per group, 10 in a, b, 8 in c, 5 in d, i, 4 in e, k, 3 in j. All results in figure were representative of 2 independent experiments with similar findings. *p < 0.05, **p < 0.01, ***p < 0.001

Parallelly increased expression of SNHG16 and TLR4 was found in human and mouse AH lung tissues. Down-regulated TLR4 levels were shown in miR-146a-overexpressed MLE-12 cells as comp