Down-regulated miR-146a expression with increased neutrophil extracellular traps and apoptosis formation in autoimmune-mediated diffuse alveolar hemorrhage

Down-regulated miR-146a expression in PBNs from DAH patients

At first, PBMCs from SLE patients and HCs were examined for miR-146a expression. Lower levels were found in SLE patients than HCs (Fig. 1a, left, 51.4 ± 7.3% versus 100.0 ± 12.4%, p < 0.001). DAH patients had lower miR-146a levels than LN patients, Nil patients or HCs (Fig. 1a, middle, p = 0.026 for LN, p = 0.002 for Nil or HCs). A negative correlation was found between miR-146a levels and activity scores (Fig. 1a, right, r = − 0.334, p = 0.009).

Fig. 1figure 1

Down-regulated miR-146a expression in PBNs from DAH patients. a Left, miR-146a levels in PBMCs from HCs and SLE patients. Middle, a negative correlation between miR-146a levels in PBMCs and activity scores. Right, miR-146a levels in PBMCs from HCs, Nil, LN and DAH patients. b A negative correlation between PBN counts and activity scores. Left, newly diagnosed and treatment-naïve SLE patients. Right, anti-neutrophil cytoplasmic antibody-positive SLE patients. c Left, miR-146a levels in PBNs from HCs and SLE patients. Middle, a negative correlation between miR-146a levels in PBNs and activity scores. Right, miR-146a levels in PBNs from HCs, Nil, LN and DAH patients. d Left, TRAF6 levels in PBNs from HCs and SLE patients. Middle, TRAF6 levels in PBNs from HCs, Nil, LN and DAH patients. Right, A negative correlation between TRAF6 and miR-146a levels in PBNs from SLE patients. Values are mean ± SEM. Horizontal lines are mean values. Patient numbers, n = 60 for PBMCs, n = 15 (left) and n = 20 (right) for PBN counts, n = 16 for PBNs. * p < 0.05, ** p < 0.01, *** p < 0.001

In the NCKUH hospitalized SLE cohort [2, 17], PBN counts in 15 newly diagnosed, treatment-naïve patients including 5 with DAH, were negatively correlated with activity scores (Fig. 1b, left, r = − 0.578, p = 0.029). Furthermore, in 20 anti-neutrophil cytoplasmic antibody-positive patients including 3 with DAH, their PBN counts were correlated inversely with SLEDAI-2 K scores (Fig. 1b, right, r = − 0.454, p = 0.044). These finding implied a pathogenic role of neutrophil death in the development and progression of SLE and its DAH manifestation by providing a source of autoantigens [4]. Lower miR-146a levels were found in PBNs from SLE patients than HCs (Fig. 1c, left, 54.9 ± 7.9% versus 100.0 ± 11.9%, p = 0.005). A negative correlation was found between miR-146a levels and activity scores (Fig. 1c, middle, r = − 0.589, p = 0.016). DAH patients had lower miR-146a levels than LN patients, Nil patients or HCs (Fig. 1c, right, p = 0.036 in all).

The levels of TRAF6, a miR-146a target molecule, in PBNs were higher in SLE patients than HCs (Fig. 1d, left, 151.3 ± 16.2% versus 100.0 ± 12.0%, p = 0.038). DAH patients had higher TRAF6 levels than LN patients, Nil patients or HCs (Fig. 1d, middle, p = 0.036 in all). A negative correlation was found between TRAF6 and miR-146a levels in SLE patients (Fig. 1d, right, r = − 0.616, p = 0.011).

Increased NETs formation in PBNs from DAH patients

PBNs from different patient groups and HCs were stimulated with PMA or LPS to induce NETs formation. SLE patients had higher percentages of spread NETs than age/sex-matched HCs (Fig. 2a, for PMA, 66.8 ± 3.4 versus 28.9 ± 2.8%, p < 0.001, for LPS, 79.9 ± 2.1 versus 3.8 ± 1.1%, p < 0.001). DAH patients had higher percentages of spread NETs than LN or Nil patients (Fig. 2b, DAH versus Nil, for PMA, 68.1 ± 4.6 versus 39.3 ± 3.2%, p = 0.036, for LPS, 85.4 ± 2.0 versus 42.6 ± 2.3%, p = 0.036, DAH versus LN, for PMA, 68.1 ± 4.6 versus 58.1 ± 3.8%, p = 0.143, for LPS, 85.4 ± 2.0 versus 61.9 ± 6.5%, p = 0.036). Furthermore, culture supernatants under PMA or LPS stimulation were examined for CitH3 levels. SLE patients had higher levels than HCs (Fig. 2c, for PMA 1.48 ± 0.56 versus 0.35 ± 0.11 ng/mL, p = 0.001, for LPS, 1.99 ± 0.49 versus 0.43 ± 0.16 ng/mL, p = 0.007). DAH patients had higher levels than LN or Nil patients (Fig. 2d, DAH versus Nil, for PMA, 2.36 ± 1.32 versus 0.54 ± 0.14 ng/mL, p = 0.036, for LPS, 3.03 ± 0.85 versus 0.63 ± 0.09 ng/mL, p = 0.036, DAH versus LN, for PMA, 2.36 ± 1.32 versus 0.83 ± 0.08 ng/mL, p = 0.071, for LPS, 3.03 ± 0.85 versus 1.19 ± 0.24 ng/mL, p = 0.036). These data suggested greater NETs formation in PBNs from DAH patients with lower miR-146a expression.

Fig. 2figure 2

Increased NETs formation in PBNs from DAH patients. a PBNs from SLE patients or HCs stimulated with PMA or LPS and stained with Sytox Green to detect DNAs morphology. Left, representative photographs from a HC and a SLE patient. Scale bar = 50 µm, magnification × 200. Right, quantification of NETs formation in HCs and SLE patients. b Quantification of NETs formation by PMA or LPS stimulation in DAH, LN and Nil patients. c CitH3 levels in PMA or LPS-stimulated supernatants from HCs and SLE patients. d CitH3 levels in PMA or LPS-stimulated supernatants from DAH, LN and Nil patients. Values are mean ± SEM. Horizontal lines are mean values. Patient numbers, n = 7 for SLE, n = 3 for DAH, n = 5 for LN, n = 5 for Nil. * p < 0.05, ** p < 0.01, *** p < 0.001

Down-regulated pulmonary miR-146a expression with increased NETs and apoptosis formation in DAH patients

Figure 3a shows H&E-stained lung tissues from DAH and PTX patients. Since there was down-regulated miR-146a expression with increased NETs formation in PBNs from DAH patients, we further examined their lung tissues for the expression of miR-146a, TRAF6, CitH3, PAD4, HMGB1, IL-6, IL-8, IFN-α and MX-1 (an ISG). Distinct expression of CitH3 colocalized with DNAs, in favor of NETs, was identified in DAH but not PTX lung tissues (Fig. 3b). Higher numbers of TUNEL-positive cells were found in lung tissues from DAH than PTX patients (Fig. 3c, 49.6 ± 8.1 versus 1.5 ± 0.6, p = 0.004). Pulmonary PAD4 levels were higher in lung tissues from DAH than PTX patients (Fig. 3d, 5,499.0 ± 428.9 versus 100.0 ± 39.2%, p < 0.001), suggestive of increased NETs formation. Down-regulated miR-146a and up-regulated TRAF-6 expression were found in lung tissues from DAH patients (p = 0.018 for miR-146a, p = 0.021 for TRAF-6). There were increased levels of HMGB1, IL-6, IL-8, IFN-α and MX-1 in DAH lung tissues (p = 0.003 for HMGB1, p = 0.013 for IL-6, p = 0.003 for IL-8, p = 0.004 for IFN-α, p = 0.033 for MX-1). These results implicated down-regulated miR-146a levels with increased expression of TRAF6, HMGB1, IL-6 and IL-8, and formation of NETs and apoptosis in the human DAH lungs.

Fig. 3figure 3

Down-regulated pulmonary miR-146a expression with increased NETs and apoptosis formation in DAH patients. a Representative H&E staining of lung tissues from a PTX and a DAH patient. Scale bar = 40 µm, magnification × 200. b Representative CitH3 IF staining (green) from a PTX and 3 DAH patients. Cell nuclei counterstained with Hoechst 33,258 (blue). Scale bar = 10 µm, magnification × 1000. c Left, representative TUNEL IF staining (green) from a PTX and a DAH patient. Cell nuclei counterstained with DAPI (blue). Scale bar = 25 µm, magnification × 400. Right, quantification of TUNEL-positive cell numbers in lung tissues. d Expression levels of PAD4, miR-146a, TRAF-6, HMGB1, IL-6, IL-8, IFN-α and MX-1 in lung tissues from 3 PTX and 3 DAH patients. Values are mean ± SEM. Horizontal lines are mean values. * p < 0.05, ** p < 0.01, *** p < 0.001

Reduced NETosis in miR-146a-overexpressed, SNHG16-silenced or miR-146a- silenced HL-60 cells

There were increased percentages of diffused/spread NETs morphology (Fig. 4a), and time-dependent down-regulated miR-146a and up-regulated CitH3/PAD4 expression in PMA-stimulated dHL-60 cells (Fig. 4b). In particular, miR-146a expression has been reported to be regulated by SNHG16, a pro-apoptotic lncRNA [27], and time-dependent up-regulated SNHG16 expression was found in PMA-stimulated dHL-60 cells (Fig. 4b). MiR-146a-overexpressed HL-60 cells had increased miR-146a levels (Fig. 4c, miR-146a versus miR-scr, 1605.7 ± 199.3 versus 100.0 ± 29.6%, p = 0.009). After PMA stimulation, there were lower NETs percentages, CitH3 and PAD4 levels in miR-146a-overexpressed dHL-60 cells (Fig. 4d, miR-146a versus miR-scr, for NETs, 17.0 ± 4.4 versus 42.3 ± 3.8%, p = 0.012, for CitH3, 1.85 ± 0.08 versus 3.65 ± 0.06 ng/mL, p = 0.003, for PAD4, 2.71 ± 0.06 versus 9.64 ± 0.60 ng/mL, p = 0.008).

Fig. 4figure 4

Reduced NETosis in miR-146a-overexpressed dHL-60 cells. a dHL-60 cells stimulated with PMA and stained with Sytox Green to detect DNAs morphology. Upper, Representative photographs from mock and PMA stimulation. Scale bar = 60 µm, magnification × 200. Lower, quantification of NETosis percentages with the diffused/spread NETs morphology. b MiR-146a (left), SNHG16 (middle left), CitH3 (middle right) and PAD4 (right) levels in dHL-60 cells stimulated with PMA for different times. c MiR-146a levels in LV-miR-146a-transfected HL-60 cells. d Quantification of NETosis percentages (left), CitH3 (middle) and PAD4 (right) levels in miR-146a-overexpressed dHL-60 cells under PMA stimulation. The presence of Cl-amidine as a PC. Values are mean ± SEM. All results in this figure were representative of 3 independent experiments with similar findings. * p < 0.05, ** p < 0.01, *** p < 0.001

A 2.7 kb stuffer was removed from pAll-EF1a-CasRx (Fig. 5a) for cloning crRNAs targeting SNHG16. Sorted GFP-positive CasRX-01 to -09- and CasRX-NC-transfected 293 T cells were examined for SNHG16 expression (Fig. 5b), with the highest silencing efficacy in CasRX-02-transfected cells. Furthermore, in CasRX-01-, -02-, -03-, -04- and -05-transfected cells with observed knockdown efficacy in SNHG16 expression varying from 27.9 to 70.2%, reciprocally up-regulated miR-146a expression was found in CasRX-01-, -02-, -03 and -04-, but not in -05-transfected cells (Fig. 5c, for SNHG16, CasRX-NC versus CasRX-01, 100.0 ± 4.5% versus 49.7 ± 6.3%, p < 0.001, CasRX-02, versus 29.8 ± 1.2%, p < 0.001, CasRX-03, versus 33.7 ± 0.4%, p < 0.001, CasRX-04, versus 53.4 ± 3.0%, p < 0.001, CasRX-05, versus 72.1 ± 9.0%, p = 0.041, for miR-146a, CasRX-NC versus CasRX-01, 100.0 ± 3.8% versus 151.5 ± 18.3%, p = 0.009, CasRX-02, versus 223.2 ± 24.0%, p < 0.001, CasRX-03, versus 184.7 ± 19.4% p = 0.002, CasRX-04 versus 139.6 ± 12.1% p = 0.033, CasRX-05, versus 111.6 ± 5.9%, p = 0.141). Sorted CasRX-02-transfected HL-60 cells were examined for SNHG16 and miR-146a expression (Fig. 5d, CasRX-NC versus CasRX-02, for SNHG16, 100.0 ± 1.5 versus 19.4 ± 1.6%, p < 0.001, for miR-146a, 100.0 ± 2.6 versus 357.6 ± 27.6%, p < 0.001). After PMA stimulation, decreased percentages of NETs morphology, and levels of CitH3 and PAD4 were found in sorted CasRX-02-transfected dHL-60 cells (Fig. 5e, CasRX-02 versus CasRX-NC, for NETs, 21.0 ± 5.1 versus 40.3 ± 3.8%, p = 0.038, for CitH3, 1.42 ± 0.12 versus 2.52 ± 0.06 ng/mL, p = 0.014, for PAD4, 2.98 ± 0.02 versus 5.27 ± 0.26 ng/mL, p = 0.012).

Fig. 5figure 5

Reduced NETosis in SNHG16- or miR-146a-silenced HL-60 cells. a Map of pAll-EF1a-CasRx (16,014 bp in length) with an EGFP insertion, a Cas13d domain, a 2.7 k bp stuffer and a 30 bp DR with an appended G. b Upper, flow cytometric graphs of sorted GFP-positive CasRx-transduced cell portion for qRT-PCR analyses including CasRx-01 to 09- and CasRx-NC-transfected 293 T cells. Lower, SNHG16 levels for silencing efficacy in CasRx-01 to 09- and CasRx-NC-transfected 293 T cells. c SNHG16 and miR-146a levels in CasRx-01 to 05- and CasRx-NC-transfected 293 T cells. d SNHG16 and miR-146a levels in CasRx-02-transfected HL-60 cells. e Quantification of NETs formation percentages (left), CitH3 levels (middle), and PAD4 levels (right) in SNHG16-silenced dHL-60 cells under PMA stimulation. f Quantification of NETs formation percentages (left), CitH3 levels (middle), and PAD4 levels (right) in PMA-stimulated CasRx-02-transfected dHL-60 cells in which miR-146a was silenced by creating sh-miR-146a-transfected stable transfectants. Values are mean ± SEM. All results in this figure were representative of at least 2 independent experiments with similar findings. * p < 0.05, ** p < 0.01, *** p < 0.001. DR: direct repeat, HA: human influenza hemagglutinin amino acids 98–106. NLS: nuclear localization sequence

Furthermore, we knocked down miR-146a expression in CasRX-02-transfected HL-60 cells to demonstrate that up-regulated miR-146a levels is involved in the action mechanisms of reduced NETosis in PMA-stimulated SNHG16-silenced dHL-60 cells. Stable transfectants were created in which miR-146a was silenced in sorted CRISPR-CasRX-02-transfected HL-60 cells. After stimulation of DMSO-induced differentiated status by PMA, higher percentages of NETs morphology and levels of CitH3 and PAD4, were found in sh-miR-146a-transfected than sh-luciferase-transfected dHL-60 transfectants (Fig. 5f, sh-miR-146a versus sh-luciferase, for NETs, 27.7 ± 2.0 versus 18.7 ± 1.5%, p = 0.023, for CitH3, 1.64 ± 0.03 versus 0.88 ± 0.03 ng/mL, p = 0.002, for PAD4, 3.49 ± 0.05 versus 2.28 ± 0.31 ng/mL, p = 0.032).

Collectively, these findings indicated that overexpressing miR-146a or silencing SNHG16 to increase miR-146a expression can reduce NETs formation in human promyelocytic cells.

Down-regulated miR-146a expression with increased NETs and apoptosis formation in mouse DAH lungs

DAH in a pristane-induced mouse model was demonstrated on day 14 with complete hemorrhage in 80% pristane-injected mice and 0% PBS-injected controls (Fig. 6a,  p < 0.001). There were higher neutrophil numbers, and lower RBC numbers, Hct and Hb levels in pristane-injected than control mice (Fig. 6b, for neutrophil, p = 0.018, for RBC, p = 0.008, for Hct, p = 0.007, for Hb, p = 0.006). Down-regulated pulmonary miR-146a levels were found in pristane-injected mice since day 4 (Fig. 6c, day 4, p = 0.003, day 9, p = 0.025, day 14, p = 0.046). Splenic miR-146a levels were also down-regulated in pristane-injected mice (day 14, p = 0.025). There were up-regulated pulmonary levels of IL-6 and IL-8 in pristane-injected mice (Fig. 6d, for IL-6, day 4, p = 0.001, day 9, p = 0.049, day 14, p < 0.001, for IL-8, day 4, p = 0.013). CitH3 and PAD4 levels in lung tissues were increased in pristane-injected mice (Fig. 6e, for CitH3, p = 0.002, for PAD4, p = 0.003), while distinct expression of CitH3 colocalized with DNAs, favoring NETs formation, was identified in pristane-injected but not control mice (Fig. 6f). Levels of HMGB1 and miR-146a target molecules IRAK1 and TRAF6, were increased in pristane-injected mice (Fig. 6e, for HMGB1, p = 0.001, for TRAF6, p = 0.004, for IRAK1, p = 0.017). Furthermore, TUNEL-positive apoptotic cells were found in pristane-injected but barely detected in PBS-treated lung tissues (Fig. 6g, 30.2 ± 4.8 versus 1.2 ± 0.4, p < 0.001).

Fig. 6figure 6

Down-regulated miR-146a expression with increased NETs and apoptosis formation in the mouse DAH lungs. a Right, representative gross and histopathological photographs in the mouse lungs with no and complete hemorrhage. Left, hemorrhage frequencies in saline- and pristane-injected mice. Scale bar = 100 µm, magnification × 100. b Neutrophil, RBC numbers, Hct and Hb levels in saline- and pristane-injected mice. c MiR-146a pulmonary (left) and splenic (right) levels on day 0, 4, 9 and 14 from saline- and pristane-injected mice. d IL-6 (left) and IL-8 (right) pulmonary levels on day 0, 4, 9 and 14 from saline- and pristane-injected mice. e Representative immunoblot assay (right) with signal intensity quantitation (left) of pulmonary CitH3, PAD4, HMGB1, TRAF6, IRAK1 and β-actin expression from saline- and pristane-injected mice. f Representative CitH3 IF staining (green) in lung tissues from saline- and pristane-injected mice. Cell nuclei counterstained with Hoechst 33,258 (blue). Scale bar = 12.5 µm, magnification × 800. g Left, representative TUNEL IF staining (green) in lung tissues from saline- and pristane-injected mice. Right, quantification of TUNEL-positive cell numbers in lung tissues. Cell nuclei counterstained with DAPI (blue). Scale bar = 25 µm, magnification, × 400. Values are mean ± SEM. Horizontal lines are mean values. Mouse numbers per group, 10 in a, 4 or 5 in b, 5 in c, d, 3 in e, 5 in g. All results in this figure were representative of 2 independent experiments with similar findings. * p < 0.05, ** p < 0.01, *** p < 0.001

Down-regulated miR-146a expression with increased NETs formation in mouse neutrophils

Pristane-complexed β-CD has been demonstrated to induce NETs formation in mouse neutrophils [28]. We further examined whether pristane-induced NETosis was through down-regulating miR-146a expression in mouse neutrophils. Before and after receiving pristane injection on day 0, 4, 9 and 14, neutrophils were isolated from thioglycolate-injected mice. There was down-regulated miR-146a expression since day 4 (Fig. 7a, day 4, p = 0.034, day 9, p = 0.047, day 14, p = 0.008). Neutrophils from day 0 naïve mice were cultured under the stimulation of βCD-pristane, IL-6, HMGB1 or LPS as a PC. In the presence of 13.3 μM, 133 μM βCD-pristane or 1 μg/mL LPS, there were down-regulated miR-146a expression (Fig. 7b), and increased diffused/spread NETs percentages with higher CitH3 production levels (Fig. 7c). Furthermore, miR-146a levels were down-regulated under the stimulation of 62.5 ng/mL, 125 ng/mL IL-6 or 1 μg/mL LPS (Fig. 7d). Notably, IL-8 is a well-known NETosis inducer as demonstrated by earlier experiments with the presence of IL-8 in mouse or human neutrophils cultures [29]. Under the stimulation of 300 ng/mL, 900 ng/mL HMGB1 or 5 μg/mL LPS, there were down-regulated miR-146a, up-regulated TRAF6 and IL-8 expression (Fig. 7e), and increased diffused/spread NETs percentages with higher CitH3 production levels (Fig. 7f).

Fig. 7figure 7

Down-regulated miR-146a expression with increased NETs formation in mouse neutrophils. a MiR-146a levels in thioglycolate-induced neutrophils purified from saline- and pristane-injected mice on day 0, 4, 9 and 14. b, MIR-146a levels in neutrophils stimulated with variable concentrations of βCD-pristane or 1 μg/mL LPS. c Left upper, representative photographs from neutrophil under 133 μM βCD-pristane or 1 μg/mL LPS stimulation. Scale bar = 30 µm, magnification × 400. Left lower, quantification of NETs morphology. Right, CitH3 levels in supernatants. d MiR-146a expression in neutrophils stimulated with various concentrations of IL-6 or 1 μg/mL LPS. e MIR-146a (left), TRAF6 (middle) and IL-8 (right) levels in neutrophils stimulated with variable concentrations of HMGB1 or 5 μg/mL LPS. f Left upper, representative photographs from neutrophil under 900 ng/mL HMGB1 or 5 μg/mL LPS stimulation. Scale bar = 30 µm, magnification × 400. Left lower, quantification of NETs morphology. Right, CitH3 levels in supernatants. Values are mean ± SEM. 5 mice per group in a. All results in Fig. 7 were representative of 2 independent experiments with similar findings. * p < 0.05, ** p < 0.01, *** p < 0.001

Reduced apoptosis and HMGB1 release in miR-146a-overexpressed MLE-12 cells

TLR4-expressed neutrophils have been shown to be activated by HMGB1, functioning as damage-associated molecular pattern (DAMP) molecule to induce NETs formation [30]. MiR-146a inhibits both of the Fas-mediated and p53-dependent apoptosis [8, 9], whereas βCD-pristane promotes the p53-dependent apoptosis [5]. In this study, we examined whether overexpressing miR-146a in alveolar cells could inhibit apoptosis to reduce HMGB1 release. There were increased apoptotic percentages and HMGB1 levels in MLE-12 cells culture, greater under Dox than under βCD-pristane stimulation (Fig. 8a, for apoptosis, mock versus βCD-pristane, 0 ± 0 versus 29.4 ± 5.6%, p < 0.001, or Dox, versus 52.1 ± 1.9%, p < 0.001, for HMGB1, mock versus βCD-pristane, 51.1 ± 0.4 versus 156.8 ± 7.9 pg/mL, p = 0.006, or Dox, versus 214.8 ± 27.0 pg/mL, p = 0.026). In addition, βCD-pristane-stimulated MLE-12 cells had a dose-dependent increase in apoptotic cell ratios, HMGB1 and IL-8 levels with down-regulated miR-146a and up-regulated TRAF6 and SNHG16 expression (Additional file 2: Fig. S2a to f).

Fig. 8figure 8

Reduced apoptosis and HMGB1 production in miR-146a-overexpressed alveolar cells. a Left, representative photographs of TUNEL IF staining (green) in MLE-12 cells stimulated with 400 μM βCD-pristane or 1 μM Dox. Cell nuclei counterstained with DAPI (blue). Scale bar = 30 µm, magnification × 600. Middle, quantification of TUNEL-positive cell percentages. Right, HMGB1 supernatant levels from stimulated MLE-12 cells. b Apoptotic cell ratios (left), HMGB1 supernatant levels (middle left), miR-146a (middle right) and SNHG16 (right) expression in MLE-12 cells stimulated with various concentrations of Dox. c MiR-146a expression in MLE-12 cells stimulated with various concentrations of IL-6. d Left, MiR-146a expression in LV-miR-146a-transfected MLE-12 cells. Middle left, representative photographs of annexin V IF staining (pink) in miR-scr- or miR-146a-overexpressed MLE-12 cells under 1 μM Dox stimulation. Cell nuclei counterstained with Hoechst 33258 (blue). Scale bar = 10 µm, magnification × 1000. Middle right, quantification of apoptotic cell percentages. Right, HMGB1 supernatant levels from miR-scr- or miR-146a-overexpressed MLE-12 cells under 1 μM Dox stimulation. Values are mean ± SEM. All results in this figure were representative of 3 independent experiments with similar findings. * p < 0.05, ** p < 0.01, *** p < 0.001

In Fig. 8b, Dox-stimulated MLE-12 cells had a dose-dependent increase in apoptotic cell ratios and HMGB1 production levels with down-regulated miR-146a and up-regulated SNHG16 expression. Notably, miR-146a levels in MLE-12 cells were down-regulated under the stimulation of IL-6 in a dose-dependent manner (Fig. 8c). MiR-146a-transfected MLE-12 cells had increased miR-146a levels (Fig. 8d, miR-146a versus miR-scr, 36,157.2 ± 3,090.0 versus 100.0 ± 16.2%, p < 0.001). After Dox stimulation, lower apoptotic percentages and decreased HMGB1 levels were found in miR-146a-overexpressed MLE-12 cells (miR-146a versus miR-scr, for apoptosis, 17.1 ± 2.0 versus 87.9 ± 0.6%, p < 0.001, for HMGB1, 678.4 ± 21.9 versus 1,036.0 ± 2.7 pg/mL, p = 0.004).

DAH suppressed by intra-pulmonary miR-146a delivery through reducing NETs and apoptosis formation

C57BL/6 mice receiving intra-tracheal LV-miR-146a delivery had lower complete hemorrhage frequencies than LV-miR-scr-treated controls (Fig. 9a, 12.5% versus 68.8%, p = 0.003). There were lower neutrophil numbers, and higher RBC numbers, Hct and Hb levels in LV-miR-146a-treated mice (Fig. 9b, for neutrophil, p < 0.001, for RBC, p = 0.004, for Hct, p = 0.012, for Hb, p < 0.001). Lower pulmonary TUNEL-positive cell numbers were found in LV-miR-146a-treated mice (Fig. 9c, 10.2 ± 2.1 versus 40.6 ± 5.8, p = 0.001). LV-miR-146a-treated mice had higher pulmonary miR-146a levels (Fig. 9d, miR-146a versus miR-scr, 287.0 ± 17.9 versus 100.0 ± 5.2%, p < 0.001), while no differences were found in splenic expression between two treatment groups (miR-146a versus miR-scr, 102.4 ± 12.8 versus 100.0 ± 23.9%, p = 0.933). There were decreased pulmonary levels of IL-6 and IL-8 in LV-miR-146a-treated mice (Fig. 9e, for IL-6, p = 0.020, for IL-8, p = 0.047). The pristane-induced mouse model is driven by a strong type I IFN response which can be targeted by miR-146a [5, 9]. LV-miR-146a-treated mice had lower pulmonary levels of IFN-α and MX-1 (Fig. 9e, for IFN-α, p < 0.001, for MX-1, p = 0.027). There was reduced expression of CitH3, PAD4, TRAF6 and IRAK-1 in LV-miR-146a-treated lung tissues (Fig. 9f,  p < 0.001 in all).

Fig. 9figure 9

DAH suppressed by intra-pulmonary miR-146a delivery through reducing NETs and apoptosis formation. a Left, representative gross and histopathological photographs in the lungs with no, partial and complete hemorrhage. Right, hemorrhagic frequencies of LV-miR-scr- and LV-miR-146a-treated mice. Scale bar = 100 µm, magnification, × 100. b Neutrophils, RBC numbers, Hct and Hb levels in LV-miR-scr- and LV-miR-146a-treated mice. c Left, representative TUNNEL IF staining (green) in lung tissues from LV-miR-scr- and LV-miR-146a-treated mice. Scale bar = 20 µm, magnification, × 400. Right, quantification of TUNEL-positive cell numbers in lung tissues. d MiR-146a levels in lung and spleen tissues from LV-miR-scr- and LV-miR-146a-treated mice. e Levels of IL-6, IL-8, IFN-α and MX-1 in lung tissues from LV-miR-scr- and LV-miR-146a-treated mice. f Representative immunoblot assay (right) with signal intensity quantitation (left) of pulmonary CitH3, PAD4, TRAF6, IRAK1 and β-actin levels from saline- and pristane-injected mice. Values are mean ± SEM. Horizontal lines are mean values. Mouse numbers per group, 16 in a, b, 5 in c, 8 in d, 8 in e, 4 in f. All results in this figure were representative of 2 independent experiments with similar findings. * p < 0.05, ** p < 0.01, *** p < 0.001

Figure 10 is a schematic representation summarizing the above experimental results. Under increased pulmonary IL-6 expression, down-regulated miR-146a levels in alveolar cells can induce cell apoptosis with the release of HMGB1, functioning as DAMP molecule to engage TLR4-expressed neutrophils, followed by activated protein kinase C (PKC) to mobilize intracellular Ca2 + and promote PAD4 activation, leading to NETs formation. Furthermore, through down-regulating miR-146a levels by IL-6 stimulation, up-regulated expression of the target molecule TRAF6 in alveolar cells and neutrophils can enhance the secretion of IL-8, a well-known inducer of NETosis.

Fig. 10figure 10

A schematic representation summarizing the experimental results. Under increased pulmonary IL-6 expression, down-regulated miR-146a expression in alveolar cells can induce cell apoptosis with the release of HMGB1, functioning as DAMP molecule to engage TLR4-expressed neutrophils, followed by activated PKC to mobilize intracellular Ca2 + and promote PAD4 activation, leading to NETs formation. Furthermore, through down-regulating miR-146a levels by IL-6 stimulation, up-regulated expression of the target molecule TRAF6 in alveolar cells and neutrophils can enhance the secretion of IL-8, a well-known inducer of NETosis

Up-regulated renal miR-146a expression in LN patients and a mouse model

Renal involvement is a common disease morbidity in SLE [3]. LN patients had lower miR-146a levels in PBMCs than those without renal involvement or HCs (Additional file 3: Fig. S3a, left, LN versus Nil, 37.6 ± 7.2 versus 79.1 ± 15.2%, p = 0.003, or HCs, versus 100.0 ± 12.4%, p < 0.001), and there was a negative correlation between miR-146a levels and daily proteinuria amounts in SLE (Additional file 3: Fig. S3a, right, r = − 0.364, p = 0.004). Nevertheless, miR-146a levels in USCs were higher in LN patients than no renal involvement or HCs (Additional file 3: Fig. S3b, LN versus Nil, 570.6 ± 204.7 versus 138.3 ± 82.3%, p = 0.002, or HCs, versus 100.0 ± 26.9%, p = 0.031).

At month 6 after pristane injection, there were hyper-cellularity and mesangial expansion in renal glomeruli (Additional file 3: Fig. S3c). At month 5 and 6, increased proteinuria levels were found in pristane-injected mice (100% incidence, Additional file 3: Fig. S3c, left, month 5, p = 0.028, month 6, p = 0.002). In addition, higher anti-dsDNA titers were noted at month 5 and 6 (100% incidence, Additional file 3: Fig. S3c, right, month 5, p = 0.006, month 6, p < 0.001). Upon sacrifice, the kidneys were removed for examining miR-146a expression before and after pristane injection and at month 0, 1, 3, 5 and 6 (Additional file 3: Fig. S3d). During the earlier GN development stage at month 1, there were down-regulated miR-146a expression, similar to the results of pristane-injected lung tissues on day 14, and up-regulated levels were found at month 5 and 6 (pristane versus saline, for month 1, 65.9 ± 10.3 versus 99.3 ± 9.7%, p = 0.041, for month 3, 106.3 ± 5.6 versus 98.7 ± 8.1%, p = 0.226, for month 5, 220.6 ± 39.5 versus 98.1 ± 17.7%, p = 0.018, for month 6, 309.6 ± 11.4 versus 97.6 ± 12.4%, p < 0.001).

In addition, at month 5 and 6 after pristane injection, arthritis (Additional file 4: Fig. S4a) was found in two out of 12 mice (17% incidence, Additional file 4: Fig. S4b), but not in PBS-injected mice. There were no differences in renal miR-146a levels between mice with and without arthritis (with versus without, 291.9 ± 17.2 versus 259.8 ± 28.3%, p = 0.637).

From the results of miR-146a levels in PBMCs from LN patients and renal miR-146a expression in the earlier GN development stage of pristane-induced mice, down-regulated miR-146a expression might contribute to a part of the LN pathogenesis. Nevertheless, in contrast to down-regulated miR-146a levels in the DAH-related pulmonary specimens, our experiments demonstrated up-regulated expression in the established GN-associated renal samples. Indeed, the expression levels of miRNAs are highly responsive to the distinct kinetics of cytokine profiles in different target organs of SLE patients [26]. Based on above findings, besides a therapeutic potential of earlier intra-renal miR-146a delivery, medications modifying the specific intra-renal cytokine milieu might provide the beneficial efficacy in LN patients.

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