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

Increasing evidences have suggested an important role of microRNAs (miRNAs) in regulating cell death processes including NETosis and apoptosis. Dysregulated expression of miRNAs and increased formation of neutrophil extracellular traps (NETs) and apoptosis participate in autoimmune-mediated diffuse alveolar hemorrhage (DAH), mostly associated with pulmonary capillaritis in systemic lupus erythematosus (SLE) patients. In particular, besides the inhibition of apoptosis, miR-146a can control innate and acquired immune responses, and regulate the toll-like receptor pathway through targeting TRAF6 to reduce the expression of pro-inflammatory cytokines/chemokines like IL-8, a NETosis inducer. Expression of miR-146a, TRAF6 and NETs were examined in peripheral blood neutrophils (PBNs) and lung tissues from SLE-associated DAH patients, and in neutrophils and pristane-induced DAH lung tissues from C57BL/6 mice. To assess NETs formation, we examined NETosis-related DNAs morphology and crucial mediators including protein arginine deiminase 4 and citrullinated Histone 3. Expression of miR-146a and its endogenous RNA SNHG16 were studied in HL-60 promyelocytic cells and MLE-12 alveolar cells during NETosis and apoptosis processes, respectively. MiR-146a-overexpressed and CRISPR-Cas13d-mediated SNHG16-silenced HL-60 cells were investigated for NETosis. MiR-146a-overexpressed MLE-12 cells were analyzed for apoptosis. Pristane-injected mice received intra-pulmonary miR-146a delivery to evaluate therapeutic efficacy in DAH. In DAH patients, there were down-regulated miR-146a levels with increased TRAF6 expression and PMA/LPS-induced NETosis in PBNs, and down-regulated miR-146a levels with increased TRAF6, high-mobility group box 1 (HMGB1), IL-8, NETs and apoptosis expression in lung tissues. HMGB1-stimulated mouse neutrophils had down-regulated miR-146a levels with increased TRAF6, IL-8 and NETs expression. PMA-stimulated HL-60 cells had down-regulated miR-146a levels with enhanced NETosis. MiR-146a-overexpressed or SNHG16-silenced HL-60 cells showed reduced NETosis. Apoptotic MLE-12 cells had down-regulated miR-146a expression and increased HMGB1 release, while miR-146a-overexpressed MLE-12 cells showed reduced apoptosis and HMGB1 production. There were down-regulated miR-146a levels with increased TRAF6, HMGB1, IL-8, NETs and apoptosis expression in mouse DAH lung tissues. Intra-pulmonary miR-146a delivery could suppress DAH by reducing TRAF6, IL-8, NETs and apoptosis expression. Our results demonstrate firstly down-regulated pulmonary miR-146a levels with increased TRAF6 and IL-8 expression and NETs and apoptosis formation in autoimmune-mediated DAH, and implicate a therapeutic potential of intra-pulmonary miR-146a delivery.


Background
Diffuse alveolar hemorrhage (DAH) is characterized by diffuse infiltrates with bleeding in alveoli, leading to respiratory failure [1]. Most of autoimmune-mediated DAH is caused by systemic lupus erythematosus (SLE)-associated pulmonary capillaritis with neutrophilic capillary infiltration and immunoglobulin/C3 vascular deposition [1,2]. SLE is a disease with overproduced autoantibodies due to a loss of immune tolerance [3]. Accelerated cell apoptosis and inefficient clearance results in excessive nuclear autoantigens, followed by immune complexes (ICs) formation with visceral deposition, causing DAH and glomerulonephritis (GN) [3,4]. A dysregulated neutrophil death, described as NETosis, has been identified in SLE [4]. Overactivated neutrophils with neutrophil extracellular traps (NETs) formation and impaired removal, contributes to disease development and progression by providing autoantigens and increasing proinflammatory responses. Enhanced apoptosis and NETs formation have been demonstrated in the SLE-mediated DAH lungs [5,6].
Based on above findings, we speculated that readjustment of dysregulated pulmonary miRNAs to reduce apoptosis and NETosis has a therapeutic potential in autoimmune-mediated DAH. In this study, expression of miR-146a, TRAF6 and NETs were examined in peripheral blood neutrophils (PBNs) and lung tissues from DAH patients, and in neutrophils and pristane-induced DAH lung tissues from C57BL/6 mice. Expression of miR-146a and its endogenous RNA SNHG16 were studied in HL-60 promyelocytic and MLE-12 alveolar cells during NETosis and apoptosis, respectively. MiR-146a-overexpressed and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas13d-mediated SNHG16-silenced HL-60 cells were investigated for NETosis. MiR-146a-overexpressed MLE-12 cells were analyzed for apoptosis. Pristane-injected mice received intra-pulmonary miR-146a delivery to evaluate therapeutic efficacy in DAH. Renal miR-146a expression was examined in lupus nephritis (LN) patients and a Balb/c mouse model. 1.5 g/dL, and histopathological or other clinical evidences [17]. LN was diagnosed by histopathological and/or laboratory findings [18]. Six patients were complicated with DAH, 5 females aged from 21 to 54 years (33.5 ± 4.8), and their age/sex-matched control groups included HCs, LN patients without DAH (LN group), and patients without DAH or LN (Nil group). DAH patients had higher SLE-DAI-2 K scores than other groups, 21.0 ± 2.3 in DAH, 9.0 ± 1.0 in LN and 2.3 ± 0.6 in Nil (DAH versus LN or Nil, p = 0.002). Venous blood and fresh urine samples were collected from patients and HCs. Surgically biopsied lung specimens were obtained from 3 DAH patients with control pulmonary tissues from 3 non-inflammatory pneumothorax (PTX) patients.

Purification of human or mouse cells
Human peripheral blood mononuclear cells (PBMCs) were purified from anticoagulated samples by Ficoll-Paque PLUS (GE-Healthcare, Chicago, IL, USA), and urine sediment cells (USCs) were collected from fresh specimens by centrifugation at 3000g for 30 min at 4 °C. PBNs were isolated by layering 5 mL anticoagulated blood over 5 mL Polymorphprep ™ (AXIS-SHIELD PoC AS, Oslo, Norway) and centrifugation at 500g for 35 min at room temperature (RT). The granulocyte fraction was harvested, and red blood cells (RBCs) were removed through hypotonic lysis. Eight-week-old female C57BL/6 Jackson National Applied Research Laboratories (C57BL/6JNarl) mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan), and housed under specific pathogen-free conditions with free access to food and water on a 12 h/12 h light-dark cycle at the NCKU Laboratory Animal Center. Animal experiments were approved by the NCKU Institutional Animal Care and Use Committee, and performed according to its guidelines. They were intraperitoneally injected with 5 mL 3% thioglycollate medium (Difco, Detroit, MI, USA), and received 5 mL phosphate-buffered saline (PBS) as lavage fluid 24 h later [19]. Peritoneal exudate cells were collected by centrifugation at 200g for 10 min at RT. Neutrophils were further isolated by Percoll gradient solution (Sigma-Aldrich, St. Louis, MO, USA). The purity of human or mouse neutrophils in this study was up to 95%. MiR-146a-overexpressed or SNHG16-silenecd GFP-positive cells with the expression of green fluorescence were sorted by a Moflo XDP cell sorter (Beckman Coulter, Mountain View, CA, USA).

Generation of lentivirus (LV) vectors carrying miR-146a
The pre-microRNA expression construct containing pre-miR-146a (System Biosciences, Palo Alto, CA, USA) is a LV-based vector in which the miR-146a precursor molecule is cloned downstream of a cytomegalovirus promoter and carrying the reported gene copGFP. Recombinant LV vectors were produced by transfecting 293 T cells (American Type Culture Collection, ATCC, Manassas, VA, USA) with pre-miR-146a or pre-miRNA scramble negative control (NC) lentivector (System Biosciences), along with packaging plasmid psPAX2 and envelope plasmid pMD2.G under the calcium phosphate precipitation [20]. LV-miR-146a or LV-miR-scrambled NC (LV-miR-scr) vectors were harvested and concentrated by ultracentrifugation. Viral titers were determined in transduction unit (TU).

qRT-PCR analyses
Total RNAs from mouse tissues and human or mouse cells were extracted by TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNAs from formalin-fixed, paraffin-embedded human tissues were purified by RNeasy FFPE Kit (Qiagen, Hilden, Germany). RNAs were reverse transcribed into complementary DNAs (cDNAs) by TaqMan Reverse Transcription Reagent Kit (Applied Biosystems, Foster City, CA, USA). cDNAs were used for qPCR by using the SYBR qPCR Mix Kit (TOOLS), and the amplification was performed in a RT-PCR system

Preparation of hydrophilic pristane
Although to study in vitro biological responses of pristane is prohibited by its hydrophobicity, this barrier can be removed by forming a hydrophilic inclusion complex with β-cyclodextrin (βCD), a D-glucose oligomer with hydrophilic surface, to effectively deliver pristane in vitro [22]. A bolus of 2 mM pristane (Sigma-Aldrich) was pipetted into 4 mM solution of βCD (Sigma-Aldrich) and stirred at RT for 4 d [5]. The crystalline complexes which precipitated out of solution were washed twice in PBS and stored at 4 °C [22]. The concentrations were measured by UV spectrometry with optical densities at 254 nm.

NETs formation in human or mouse cells
PBNs (5 × 10 5 cells/mL) were allowed to adhere to poly-L-lysine (Sigma-Aldrich) coated 24-well plate in the presence of 250 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) or 3 μg/mL lipopolysaccharide (LPS) from Pseudomonas aeruginosa 10 (Sigma-Aldrich) [23] for 4 h under 37 °C. Mouse neutrophils (10 6 cells/ mL) were cultured in 3.5 cm dish for 30 min under 37 °C. The attached neutrophils were incubated under the same condition with different concentrations of βCDpristane or mouse high-mobility group box 1 (HMGB1, Atlantis Bioscience, Singapore, Republic of Singapore) for 4 h under 37 °C, and with the LPS stimulation as a positive control (PC). Human promyelocytic cells (HL-60, ATCC) were cultured with 10 6 cells/mL in 3.5 cm dish in the presence of 1.25% dimethyl sulfoxide (DMSO, Sigma-Aldrich) for 5 d under 37 °C to induce differentiated HL-60 (dHL-60) cells. These cells were further cultured in the presence of 50 ng/mL PMA with serum-free X-VIVO 15 medium (Lonza, Basel, Switzerland) for 4 h under 37 °C. After culture, human or mouse cells were stained with Sytox Green (Thermo Fisher Scientific, Waltham, MA, USA) to detect DNAs under fluorescence microscopy. Their morphology was categorized into lobulated neutrophils, de-lobulated neutrophils, diffused NETs or spread NETs category [24]. These cells were further subjected to qRT-PCR analyses. Their culture supernatants and cells lysates were quantified by enzyme-linked immunosorbent assay (ELISA) for the levels of CitH3 and PAD4 (Cayman, Ann Arbor, Michigan, USA), respectively.
NETs formation in miR-146a-overexpressed, SNHG16-silenced or miR-146a-silenced HL-60 cells HL-60 cells (10 6 cells/mL) in 3.5 cm dish were transfected with LV-miR-146a, LV-miR-scr, CRISPR-CasRX-SNHG16 or CRISPR-CasRX-NC for 48 h under 37 °C in the presence of polybrene. GFP-positive cells were sorted and examined by qRT-PCR analyses. Sorted or mock cells were cultured in the presence of 1.25% DMSO for 5 d to induce dHL-60 cells. In addition, miR-146a #1or luciferase-silenced CRISPR-CasRX-02-transfected HL-60 stable transfectants were cultured in the same condition for 5 d. These cells were then stimulated with 50 ng/mL PMA under serum-free X-VIVO 15 medium for 4 h, stained with Sytox Green, and observed under fluorescence microscopy. Their culture supernatants and cells lysates were examined for CitH3 and PAD4 levels, respectively. Un-transfected dHL-60 cells were stimulated in the presence of 100 μM chloramidine (Cl-amidine, Cayman), a PADs inhibitor to inhibit NETosis as a PC.

Apoptosis induction in MLE-12 cells
Mouse alveolar cells (MLE-12, ATCC) were seeded with 1 × 10 6 cells/mL in 3.5 cm dish in the presence of different concentrations of doxorubicin (Dox, TTY Biopharm, Taipei, Taiwan), a DNA damage inducer to trigger p53-dependent cell apoptosis [25], or different concentrations of βCD-pristane for 24 h under 37 °C. After stimulation, these cells were subjected to qRT-PCR analyses. Their culture supernatants were assessed for HMGB1 concentrations by ELISA (LSBio, Seattle, WA, USA). After stimulation, cells were stained with PE-Annexin V (BD Pharmingen, San Diego, CA, USA) and 7-aminoactinomycin D (7-AAD, BD Pharmingen). Annexin V-positive and 7-AAD-negative cells were defined as apoptosis, and average apoptotic percentages without stimulation was defined as apoptotic cell ratios 1.0 [5,26]. Alternatively, these cells were stained by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) detection cocktail (Promega, Madison, WI, USA) with cell nuclei counterstained by DAPI (Sigma-Aldrich), and observed under confocal microscopy with a FluoView FV3000 (Olympus, Tokyo, Japan).

Apoptosis induction in miR-146a-overexpressed MLE-12 cells
MLE-12 cells (1 × 10 6 cells/mL) in 3.5 cm dish were transfected with LV-miR-scr or LV-miR-146a for 48 h under 37 °C in the presence of polybrene. GFP-positive cells were sorted and examined by qRT-PCR analyses. Sorted cells were stimulated with 1 µM Dox for 24 h under 37 °C. After stimulation, these cells were incubated with Hoechst 33,258 (Thermo Fisher Scientific) and Annexin V Alexa Fluor 647 conjugate (BioLegend, San Diego, CA, USA), and observed under confocal microscopy. Culture supernatants were examined for HMGB1 levels. In addition, un-transfected MLE-12 cells were stimulated in the presence of 10 µM Z-VAD-FMK (Selleckchem, Houston, TX, USA), a pan-caspase inhibitor to inhibit apoptosis as a PC.

Pristane-induced mouse DAH or LN model
Eight-week-old female C57BL/6JNarl mice received intraperitoneal injection of 0.5 mL pristane to induce DAH, and their controls were injected with 0.5 mL PBS  [5]. They were sacrificed on day 0, 4, 9 and 14 to obtain their lungs and spleen. Anticoagulated blood samples were measured for neutrophil, RBC numbers, hematocrit (Hct) and hemoglobulin (Hb) levels by a blood cell analyzer (Scil Vet Focus 5, Scil Animal Care, Viernheim, Germany). Eight-week-old female BALB/cJNarl mice were purchased from the National Laboratory Animal Center, and received intraperitoneal injection of 0.5 mL pristane to induce LN, while their controls were injected with 0.5 mL PBS [26]. Urine specimens were collected for measuring proteinuria (protein/creatinine) by test strips (Arkray, Edina, MN, USA). The results were determined by semiautomated urine chemistry analyzer (Arkray RT-4010) at month 0, 1, 3, 5 and 6. Blood samples from mice were examined for the presence of anti-dsDNA levels with an ELISA kit (Alpha Diagnosis, San Antonio, TX, USA) at month 0, 1, 3, 5 and 6. Their kidneys were removed for measuring miR-146a levels at month 0, 1, 3, 5, and 6, and for analyzing renal histopathology at month 6.

Intra-tracheal LV-miR-146a delivery and therapeutic evaluation
Mice received 2 × 10 9 TU/mL of LV-miR-146a or LV-miR-scr by intra-tracheal delivery of fluid bolus into the posterior oropharynx above the tracheal entrance [5], and intraperitoneal injection of 0.5 mL pristane. DAH was evaluated according to gross and histopathological findings with three categories including no, partial and complete hemorrhage on day 14 [5].

Histopathological, TUNEL and immunofluorescence staining
Removed lung and kidney tissues were fixed in 10% buffered formalin overnight, and embedded in paraffin. Lung tissues were cut into 5 µm sections, and stained with hematoxylin and eosin (H&E). Paraffin-embedded sections were de-paraffinized in xylene, dehydrated in ethanol and rehydrated in distilled water. To determine GN, mouse renal tissues were analyzed by Periodic acid-Schiff (PAS) staining [26]. For TUNEL staining, de-paraffinized lung sections were treated by proteinase K to reactivate antigens, re-fixed by 4% formaldehyde, incubated with equilibrate buffer, and finally labelled by TUNEL detection cocktail [5,26]. TUNEL-positive cells were determined by averaging the number from 3 fields (× 400) of positively stained cells with the highest density in each section. Cell nuclei were counterstained with DAPI. Fluorescence was detected by confocal microscopy. For detecting the expression of CitH3, de-paraffinized human or mouse lung sections were stained with anti-CitH3 antibodies, followed by Alexa Fluor 488-conjugated antibodies (Thermo Fisher Scientific). Cell nuclei were counterstained with Hoechst 33258. Fluorescence was detected by confocal microscopy.

Statistical analyses
Data are expressed as the mean ± standard error of mean (SEM). MiR-146a levels, TRAF6 levels, morphology percentages or CitH3 concentrations between patients and HCs or different patient groups were analyzed by Mann-Whitney U test. Correlation analysis was performed by Spearman correlation coefficient test. Complete hemorrhage frequencies between LV-miR-scr-and LV-miR-146a-treated mice were compared by Fisher's exact test. Differences in other analyses were determined by Student's t test. p values less than 0.05 were considered significant in this study with symbols as * for p < 0.05, ** p < 0.01 and *** for p < 0.001.

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

Discussion
In this study, down-regulated and up-regulated miR-146a levels were found in PBMCs and USCs from LN, respectively. Down-regulated miR-146a levels in PBMCs has been demonstrated in SLE, negatively associated with increased IL-6/IL-8 and TRAF6 expression, particularly in renal involvement; however, miR-146a levels were up-regulated in USCs and glomerular tissues from LN [9,31]. Notably, there was predominant expression of Th1 cytokines, IFN-γ/IL-2, in USCs from LN [32]. Since cytokines can induce cellular miRNAs expression [9,26], conflicting findings between miR-146a levels in PBMCs and USCs might reflect inconsistent circulating and renal cytokine profiles in LN. In our experiments, DAH lung tissues had higher IL-6 but lower miR-146a expression. MiR-146a levels in neutrophils or alveolar cells culture were down-regulated by the IL-6 stimulation. MiRNAs can be epigenetically regulated by IL-6, while miR-146a promoter has CpG island with putative STAT-1/NF-κB binding sites [31]. Further studies are needed to elucidate whether down-regulated miR-146a expression in DAH lung tissues is caused by IL-6-induced DNAs methylation. MiR-146a −/− mice receiving the injection of LPS, a TLR4 agonist, had increased NETs formation [33], whereas TLR4 −/− mouse neutrophils were associated with reduced NETosis [34]. Oxidized low density lipoprotein could induce NETs formation in neutrophils via TLRs activation with IRAK/PKC/MAK pathways as signaling mediators [35]. These findings suggest that lower miR-146a levels enhance NETs formation through regulating the TLRs signaling. In this study, there was down-regulated miR-146a expression in dHL-60 cells during NETosis, while miR-146a-overexpressed dHL-60 cells had reduced NETs formation. Lower miR-146a levels in PBNs from DAH patients caused greater NETs formation with higher amounts of ICs formation/deposition, leading to severer activity than other patients. SLEassociated or pristane-induced DAH lung tissues had enhanced NETosis with down-regulated miR-146a and up-regulated TRAF6 expression, while intra-pulmonary miR-146a delivery could lessen TRAF6 levels to reduce NETs formation. Our experimental data implicated that pulmonary NETosis in DAH is regulated by miR-146a through targeting the expression of TLRs pathwaysrelated molecule TRAF6.
IL-8 is secreted by cells expressing TLRs in response to inflammatory stimuli [36]. Although neutrophils migrate when sensing the IL-8 gradient, higher concentrations can saturate the receptors and prohibit their chemotaxis, followed by NETs formation [37]. Increased NETs formation and IL-8 levels were identified in bronchoalveolar lavage fluid from pneumonia patients [38], while reduced miR-146a with higher IL-8 expression was demonstrated in biopsied asthmatic bronchial cells [39]. Despite no identification of MREs within IL-8 mRNA by existing algorithms, miR-146a can negatively regulate IL-8 production at the translational level [40]. In our experiments, the DAH lungs had down-regulated miR-146a, up-regulated TRAF6 and increased IL-8 expression with enhanced NETosis, while intra-pulmonary miR-146a delivery could suppress TRAF6 and IL-8 expression with reduced NETs formation. Besides, stimulated neutrophils and alveolar cells had down-regulated miR-146a, and upregulated TRAF6 with increased IL-8 expression. These results indicated that, in DAH, miR-146a can regulate pulmonary NETs formation by targeting TRAF6 to modulate IL-8 expression.
Pulmonary NETosis with colocalized CitH3 and DNAs, has been demonstrated in the pristane-injected lungs, while DNase-1 inhalation to clear NETs could suppress mouse DAH [28]. Reduced DAH severity was shown in mice with myeloid-specific PAD4 deletion, displaying a protective role with the loss of PAD4-dependent NETosis [6]. We identified NETs formation with down-regulated miR-146a expression in human and mouse DAH lung tissues, while intra-pulmonary miR-146a delivery could suppress DAH through reducing NETosis. Collectively, these findings suggested the involvement of miR-146a-regulated NETs formation and the potential of anti-NETs therapy in DAH patients.
HMGB1 is released from activated or damaged cells, serving as DAMP molecule to interact with TLRs-expressed cells and participate in autoimmune responses [41]. In particular, apoptotic cells are an important source of HMGB1 [42]. Circulating HMGB1 levels were positively correlated with disease activity in SLE [41]. Furthermore, pristane-injected mice released HMGB1 from apoptotic cells, activating neutrophils through the TLRs signaling to form NETs [43]. Incubation of bone marrow-derived mouse neutrophils with HMGB1 could induce NETs formation [44], while our experiments demonstrated HMGB1-induced NETosis in cultured thioglycolate-activated peritoneal mouse neutrophils. In this study, DAH lung tissues had downregulated miR-146a levels with increased apoptosis and HMGB1 expression. Stimulated alveolar cells had down-regulate miR-146a expression with increased apoptosis and HMGB1 release, while miR-146a-overexpressed cells had reduced apoptosis and HMGB1 production. Our experimental results demonstrated that increased apoptosis by down-regulated miR-146a expression in alveolar cells could enhance the HMGB1 release, engaging with TLR4-expressed neutrophils to induce pulmonary NETosis.