Pulmonary surfactant protein A and surfactant lipids upregulate IRAK-M, a negative regulator of TLR-mediated inflammation in human macrophages

Abstract

Alveolar macrophages (AMs) are exposed to frequent challenges from inhaled particulates and microbes and function as a first line of defense with a highly regulated immune response because of their unique biology as prototypic alternatively activated macrophages. Lung collectins, particularly surfactant protein A (SP-A), contribute to this activation state by fine-tuning the macrophage inflammatory response. During short-term (10 min–2 h) exposure, SP-A's regulation of human macrophage responses occurs through decreased activity of kinases required for proinflammatory cytokine production. However, AMs are continuously exposed to surfactant, and the biochemical pathways underlying long-term reduction of proinflammatory cytokine activity are not known. We investigated the molecular mechanism(s) underlying SP-A- and surfactant lipid-mediated suppression of proinflammatory cytokine production in response to Toll-like receptor (TLR) 4 (TLR4) activation over longer time periods. We found that exposure of human macrophages to SP-A for 6–24 h upregulates expression of IL-1 receptor-associated kinase M (IRAK-M), a negative regulator of TLR-mediated NF-κB activation. Exposure to Survanta, a natural bovine lung extract lacking SP-A, also enhances IRAK-M expression, but at lower magnitude and for a shorter duration than SP-A. Surfactant-mediated upregulation of IRAK-M in macrophages suppresses TLR4-mediated TNF-α and IL-6 production in response to LPS, and IRAK-M knockdown by small interfering RNA reverses this suppression. In contrast to TNF-α and IL-6, the surfactant components upregulate LPS-mediated immunoregulatory IL-10 production, an effect reversed by IRAK-M knockdown. In conclusion, these data identify an important signaling regulator in human macrophages that is used by surfactant to control the long-term alveolar inflammatory response, i.e., enhanced IRAK-M activity.

the lungs are frequently exposed to microbes and particulates. Although the majority of these are cleared by the upper airways, some reach the terminal bronchioles and alveoli. The inflammatory response in the alveolar microenvironment is tightly regulated to avoid damage to the gas-exchanging delicate structures through the concerted efforts of the innate and adaptive immune system (18, 69). Two important components of the innate immune response are alveolar macrophages (AMs) and pulmonary surfactant, the latter of which regulates macrophage function (1, 11, 12, 20). AMs are resident professional phagocytes that are involved in the clearance of invading microbes and particulates that land in the air spaces without creating excessive inflammation (39). AMs are characterized by enhanced expression and activity of certain pattern recognition receptors (PRRs), such as the mannose receptor (MR), a subset of Toll-like receptors (TLRs), scavenger receptor A, and the nuclear receptor peroxisome proliferator-activated receptor-γ (2, 32, 38, 59, 64). They are considered to be immunoregulatory cells with high phagocytic ability, low microbicidal activity, poor antigen presentation, and suppressive lymphocyte activity, an attenuated respiratory burst and production of the anti-inflammatory PGE₂ as well as TGFβ (9, 27, 28, 39, 44, 54, 61, 72). Through the increased activity of peroxisome proliferator-activated receptor-γ, they also function to inhibit the amplification of signaling leading to a robust proinflammatory response through trans-repression of the transcriptional factors NF-κB, activator protein 1, and STAT (31, 59, 60, 70). On the basis of the above-mentioned characteristic features, AMs have been recently considered to be more polarized toward an M2 phenotype, known as alternatively activated macrophages (24, 47, 51), although they represent a highly heterogeneous cell population.

Surfactant is composed of phospholipids, such as dipalmitoylphosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, sphingomyelin, phosphatidylinositol, phosphatidylserine, and four surfactant-associated proteins, including surfactant protein (SP) A (SP-A) and SP-D, which are collectins (48, 73). SP-A is secreted by type II alveolar cells and contains a collagen-like domain, a short linking domain, and a globular calcium-dependent carbohydrate recognition domain (74). It is a multimeric protein with trimeric units that form an octadecamer protein (33, 37, 71). SP-A enhances the phagocytosis of opsonized and nonopsonized particulates by regulating the expression of surface phagocytic receptors (2, 38, 67). SP-A knockout mice exhibit reduced clearance of group B streptococci with increased production of proinflammatory cytokines, such as TNF-α and IL-6 (40), and also reduced clearance of Pseudomonas aeruginosa, Haemophilus influenzae, and respiratory syncytial virus (41–43). In murine AMs, SP-A has been shown to inhibit the production of reactive nitrogen intermediates in response to Mycobacterium tuberculosis infection (55). Replacement of SP-A by exogenous treatment improves the clearance of microorganisms (40). We previously showed that incubation of SP-A with human macrophages regulates the expression and activity of the PRR TLR2, but not TLR4 (26). Short-term (10 min–2 h) exposure of SP-A to human macrophages decreases TLR-mediated TNF-α production by inhibiting the activity of signaling kinases (Akt and MAPKs) required for NF-κB transcriptional activation. Although these studies model the initial exposure of macrophages to SP-A, they do not account for the fact that AMs are constantly exposed to surfactant in the alveolus.

Regulation of macrophage immune responses over time is achieved by various mechanisms, including the upregulation of negative regulators of inflammation, such as IL-1 receptor-associated M (IRAK-M), suppressor of cytokine signaling (SOCS-1 and SOCS-3), and Toll-interacting protein, which block signaling pathways, resulting in decreased production of inflammatory mediators (7, 35, 76). Among these, IRAK-M plays a critical role in the regulation of innate immunity. It is ubiquitously expressed in monocytes and macrophages and suppresses TLR-mediated NF-κB activation by preventing the activation of TNF receptor-associated factor (TRAF6) (35). IRAK-M knockout mice are relatively resistant to bacterial infection (16), and IRAK-M-deficient macrophages exhibit increased production of inflammatory cytokines in response to TLR/IL-1 stimulation and bacterial challenge (35). IRAK-M expression is enhanced by TLR ligands (35). In addition, IRAK-M can suppress sepsis-induced lung innate immunity (16), is involved in peptidoglycan-induced tolerance in macrophages (53), and negatively regulates the production of TLR-dependent IL-12 p40 (56).

Here we identify and characterize the longer-term inhibitory effects of SP-A and Survanta on the TLR-4-mediated inflammatory response of human macrophages. We demonstrate that exposure of macrophages to SP-A, Survanta, or both upregulates the expression of IRAK-M over 24 h. Most importantly, upregulation of IRAK-M leads to decreased production of LPS-mediated proinflammatory cytokine responses such as TNF-α and IL-6. Knockdown of IRAK-M in human macrophages reverses the inhibitory effect of SP-A and Survanta. Besides their effect on IRAK-M expression, exposure to SP-A and Survanta leads to an increase in immunoregulatory IL-10 production following LPS stimulation. Together, these studies provide new insight into mechanisms underlying the immunoregulatory effects of surfactant on human macrophages.

MATERIALS AND METHODS

Reagents and antibodies.

RPMI 1640 medium with l-glutamine (RPMI) and HEPES buffer were purchased from GIBCO-Invitrogen (Invitrogen Life Technologies, Carlsbad, CA); human serum albumin from CSL Behring (Kankakee, IL); BSA, LPS, and naphthol blue black from Sigma Aldrich (St. Louis, MO); Survanta from Hospira (Lake Forest, IL); antibody specific for IRAK-M from Cell Signaling Technology (Boston, MA); β-actin antibody from Santa Cruz Biotechnology (Santa Cruz, CA); and Accell IRAK-M small interfering RNA (siRNA) and scramble siRNA from Thermo Scientific Dharmacon RNAi Technologies (Chicago, IL).

SP-A purification.

SP-A was purified as previously described (26). Briefly, bronchoalveolar lavage (BAL) from alveolar proteinosis patients was centrifuged at 20,000 g, washed repeatedly, and eluted using 2 mM EDTA at 4°C. SP-A was purified from the supernatant by separation over a mannose-Sepharose affinity chromatography column, and EDTA was removed by dialysis against 10 mM HEPES + 25 mM NaCl. All steps were performed under sterile conditions at 4°C whenever possible. Endotoxin-free water (Baxter, Round Lake, IL) was used for all steps to reduce LPS contamination. Purified protein was examined by SDS-PAGE and Western blot using rabbit anti-SP-A antiserum, and endotoxin contamination was determined using a Limulus amoebocyte lysate assay kit with Escherichia coli LPS standards (Bio-Whittaker/Cambrex, East Rutherford, NJ). Endotoxin concentration was ≤0.25 pg/μg protein.

Isolation and culture of human monocyte-derived macrophages and AMs.

Monocyte-derived macrophage (MDM) monolayers were prepared from healthy, purified protein derivative-negative human volunteers according to a protocol approved by the Institutional Review Board of The Ohio State University, as described elsewhere (29). Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood on a Ficoll cushion and then cultured in Teflon wells (Savillex, Minnetonka, MN) for 5 days in the presence of 20% autologous serum. The wells were placed on ice for 30 min, and the PBMCs were removed by washing. MDMs in the cultured PBMCs were adhered to 12- or 24-well tissue culture plates (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ) for 2–3 h at 37°C in 5% CO₂ in 10% autologous serum. Lymphocytes were then washed away, and MDM monolayers were repleted with RPMI containing 10% autologous serum and incubated overnight before they were used for experiments. The conditions for MDM transfection experiments and confocal microscopy studies are described below.

Human AMs were isolated from BAL of healthy human donors, as previously described (21), according to a protocol approved by the Institutional Review Board at The Ohio State University. Briefly, the BAL was centrifuged (200 g, 4°C for 10 min), the supernatant was removed, and the pellet was resuspended in RPMI and washed twice with RPMI. AMs were plated in tissue culture plates with 10% human serum supplemented with penicillin (1,000 U/ml), incubated in a CO₂ incubator at 37°C for 2 h, washed with warm RPMI to remove nonadherent cells and penicillin, and used for experiments.

Macrophage stimulation, cell lysis, and Western blotting.

Day 5 MDMs or AMs were adhered in a 12-well tissue culture plate for 2 h at 37°C, washed, and repleted with 10% autologous serum for 24 h. MDMs were incubated with SP-A (10 μg/ml), Survanta (100 μg/ml), or SP-A (10 μg/ml) + Survanta (100 μg/ml) in 1% serum RPMI for 1, 6, 12, or 24 h. These were found to be optimal concentrations in preliminary experiments. The monolayers were washed once with PBS and lysed in TN-1 lysis buffer [50 mM Tris (pH 8.0), 10 mM EDTA, 10 mM Na₄PO₇, 10 mM NaF, 1% Triton X-100, 125 mM NaCl, 10 mM Na₃VO₄, 10 μg/ml aprotinin, and 10 μg/ml leupeptin], incubated on ice for 10 min, and centrifuged at 17,949 g for 10 min at 4°C to remove cell debris (59). Protein concentrations of the cleared cell lysates were measured using the Pierce bicinchoninic acid protein assay kit (Thermo Scientific). Cell lysates were separated by SDS-PAGE under reduced and denaturing conditions and analyzed by Western blot by probing with the antibody of interest and development using enhanced chemiluminescence (Amersham Biosciences, Pittsburg, PA). The protein band intensities were measured using National Institutes of Health ImageJ software. Background intensity was subtracted from each sample and then normalized to β-actin. Percent increase was determined as follows: treated sample band intensity − untreated sample band intensity ÷ untreated sample band intensity × 100.

Transfection of MDMs.

MDMs were transfected with scramble siRNA or IRAK-M siRNA (target sequence GUAGAGUAGUGUUAGAUGA) at 100 nmol each using Nucleofector (Amaxa Biosystems, Gaithersburg, MD). Briefly, day 5 PBMCs (1 × 10⁷) were resuspended in 100 μl of Nucleofector solution; then scramble siRNA or IRAK-M-specific siRNA was added, and the cells were incubated at room temperature for 5 min and subjected to nucleofection according to the manufacturer's instructions. PBMCs were then seeded on 12-well tissue culture plates (except for confocal microscopy studies, see below) with 1.0 ml of RPMI supplemented with 10% autologous serum and incubated for 3 h at 37°C in 5% CO₂. After 3 h, adhered transfected cells (MDMs) were washed and repleted with warm RPMI containing 10% autologous serum overnight.

To ensure that the same number of cells were being studied in the scramble siRNA and IRAK-M-specific siRNA groups, we performed a cell enumeration assay, as described previously (59). Briefly, scramble siRNA- and IRAK-M siRNA-transfected MDMs were plated in 24-well plates; after 2 h, the cells were washed and repleted with 20% autologous serum for 24 h. Cells were then washed once with PBS and lysed with 1% cetavlon in 0.1 M citric acid with 0.05% naphthol blue black, pH 2.2, for 15 min at room temperature. Cell lysates were loaded on a hemocytometer, and stained nuclei representing cells were enumerated using phase contrast microscopy (52).

Cytokine assays.

Day 5 MDMs or MDMs transfected with scramble siRNA or IRAK-M siRNA (4 × 10⁵) were incubated with SP-A (10 μg/ml), Survanta (100 μg/ml), or SP-A (10 μg/ml) + Survanta (100 μg/ml) in 1% RPMI for 12 or 24 h and then treated with LPS (100 ng/ml) for 5 h. After 5 h, cell-free culture supernatants were collected and used to measure TNF-α or IL-6 by ELISA (R & D Systems).

RNA isolation and gene expression studies by quantitative RT-PCR.

MDMs were incubated with SP-A (10 μg/ml) or Survanta (100 μg/ml) in 1% serum RPMI for 1, 6, 12, or 24 h. At each time point, MDMs were washed with PBS and lysed in TRIzol, and total RNA was isolated using the Qiagen RNeasy column method. The Experion automated electrophoresis system (Bio-Rad, Hercules, CA) was used to determine the quality and quantity of RNA samples. The total RNA (100 ng) was reverse-transcribed to cDNA by using reverse transcriptase enzyme (SuperScript II, Invitrogen), and quantitative RT-PCR (qRT-PCR) was performed using a human IRAK-M TaqMan gene expression kit (Applied Biosystems, Carlsbad, CA) and Bio-Rad CFX96 Real Time System. Negative controls included no reverse transcriptase and no template (cDNA) in the reactions. The software provided by Bio-Rad was used to normalize IRAK-M gene expression to β-actin as a housekeeping gene. Percent increase was calculated from expression values relative to untreated. Triplicate samples were analyzed in duplicate by qRT-PCR.

Analysis of IRAK-M expression in MDMs by confocal microscopy.

MDMs (2 × 10⁵) were adhered to glass coverslips in 24-well tissue culture plates for 2 h at 37°C and washed to remove lymphocytes. The cells were washed, fixed with 2% paraformaldehyde (20 min at room temperature), permeabilized by treatment with 100% methanol for 1 min, washed, and incubated for 3 h in 10% nonfat milk in PBS as the blocking reagent. The cells were then incubated with IRAK-M (1.0 μg/ml) or the appropriate isotype (1.0 μg/ml) control antibody overnight at 4°C in 1% BSA/PBS-Tween 20. Then the cells were washed with 1% BSA/PBS-Tween 20 and counterstained with an Alexa Fluor 488-conjugated secondary antibody (1:500 dilution) for 1 h at room temperature. Nuclei were labeled with 0.1 μg/ml of the DNA stain 4′,6-diamidino-2-phenylindole (Invitrogen/Molecular Probes) in PBS for 5 min at room temperature. The cells were then washed with PBS, and coverslips were mounted on glass slides. Slides were viewed using a laser scanning confocal microscope (Flowview 1000, Olympus).

Statistics.

For IRAK-M expression and cytokine production analyses in MDMs, the magnitude of the response in each independent experiment varied among the donors; however, the pattern of experimental results was the same from donor to donor. To account for this variability, we normalized the data to an internal control in each experiment. A ratio of experimental results to control was obtained (or percent change was derived), and results were analyzed using one-factor ANOVA or Student's t-test where appropriate. Statistical significance was defined as P < 0.05.

RESULTS

SP-A and Survanta enhance IRAK-M expression in human macrophages.

Studies of IRAK-M expression and function have been conducted largely in murine macrophages or macrophage cell lines (16, 34, 35, 53, 56). Thus there are limited data available about IRAK-M and human macrophages and none in response to surfactant. We determined that human AMs express high levels of IRAK-M compared with MDMs isolated from healthy donors (Fig. 1A). Since AMs are exposed to surfactant components, using qRT-PCR, Western blot, and confocal microscopy, we began our investigation by determining the expression of IRAK-M mRNA and protein in human MDMs that were treated with SP-A, the most abundant surfactant collectin, or surfactant lipid in the form of Survanta for different time periods. Using qRT-PCR, we found that IRAK-M mRNA expression is significantly increased when macrophages are treated with SP-A or Survanta, especially at 6 and 12 h (Fig. 1B). In accordance with the mRNA studies, IRAK-M protein levels were increased by SP-A or Survanta (Fig. 1C). Whereas the effect of SP-A on IRAK-M protein production was sustained over 24 h, the effect of Survanta was shorter-lived (over 12 h). The results were confirmed by confocal microscopy (Fig. 1D). Given the expected metabolism of surfactant components within macrophages (13), we next examined whether a more continuous exposure to SP-A or Survanta maintains the expression of IRAK-M in human macrophages for a longer period of time. MDMs were incubated with SP-A or Survanta for 36 h, with the addition of these surfactant components at 0 h alone or at 0, 12, and 24 h (Fig. 2A), and IRAK-M mRNA levels were analyzed by qRT-PCR. Repeated addition of SP-A or Survanta prolonged the enhanced expression of IRAK-M in macrophages compared with a single dose (Fig. 2B). These results are consistent with the concept that enhanced expression of IRAK-M in AMs is due to the constant exposure to alveolar surfactant components.

Fig. 1.

Surfactant protein A (SP-A) and Survanta stimulation upregulates IL-1 receptor-associated kinase M (IRAK-M) expression in human macrophages. A: human alveolar macrophages AMs (HAM) were harvested from a healthy donor, and IRAK-M protein levels were compared with monocyte-derived macrophages (MDMs) (equal amount of protein loaded) by Western blot. IB, immunoblot. Blots are representative of 2 experiments. B: day 5 MDMs were stimulated with SP-A (10 μg/ml) or Survanta (100 μg/ml) or left unstimulated [resting (R)] for 1, 6, 12, or 24 h. Total RNA was extracted and analyzed for IRAK-M expression by quantitative real-time PCR. Values (means ± SE) are cumulative data obtained from 3 independent experiments (3 donors, each experiment performed in triplicate). P < 0.005 compared with resting for all time points by 1-factor ANOVA. C: at 1, 6, 12, and 24 h, whole cell lysates were collected and analyzed for IRAK-M protein levels by Western blot using an anti-IRAK-M antibody. Blots are representative of 3 independent experiments. Bar graph shows cumulative data for densitometry analysis of band intensities from 3 independent experiments (3 donors). Values are means ± SE. D: MDMs on coverslips were stimulated with or without SP-A (10 μg/ml) or Survanta (100 μg/ml) for 12 h. MDMs were fixed with paraformaldehyde, permeabilized, stained with anti-IRAK-M antibody and then with rabbit Alexa Fluor 488 and 4′,6-diamidino-2-phenylindole (DAPI, blue) for nuclear localization, and examined by confocal microscopy. Scale bars, 20 μm. Photomicrograph is representative of 3 experiments.

Fig. 2.

Prolonged expression of IRAK-M in macrophages with repeated dosing of SP-A or Survanta. A: schematic diagram showing addition of SP-A or Survanta over the course of the experiment and analysis (0–36 h). Closed arrows indicate time and input of SP-A (10 μg/ml) or Survanta (100 μg/ml); open arrows indicate time of cell lysis. At each time point, cells were lysed, and total RNA was extracted for analysis of IRAK-M expression by quantitative real-time PCR. B: results (means ± SD) from 2 independent experiments (2 donors) performed in triplicate.

Because of its physiological relevance, we next sought to determine whether SP-A and Survanta in combination synergize the expression of IRAK-M in human macrophages. MDMs were incubated with SP-A, Survanta, or SP-A + Survanta for 12 and 24 h, and cell lysates were used to analyze the IRAK-M protein levels. SP-A + Survanta increased IRAK-M expression in additive fashion at 12 h, with increased levels maintained at 24 h (data not shown). Together, the results provide strong evidence that surfactant components contribute to IRAK-M expression in human macrophages.

Surfactant-mediated upregulation of IRAK-M inhibits LPS-induced TNF-α and IL-6 production in human macrophages.

TLR-mediated myeloid differentiation factor 88 (MyD88) signaling leads to the recruitment of IRAK-1 and IRAK-4 and forms a complex with TRAF6, which in turn results in activation of MAPKs and NF-κB required for proinflammatory cytokine gene expression (25). This signaling pathway is tightly regulated. IRAK-M has been shown to negatively regulate TLR4-mediated cytokine production (16, 35). Thus, to examine the effect of surfactant-mediated expression of IRAK-M on cytokine production, MDMs were pretreated with or without SP-A, Survanta, or SP-A + Survanta for 12 and 24 h (to upregulate the IRAK-M expression) and subsequently stimulated with the TLR4 agonist LPS for 5 h. The cell-free culture supernatants were then analyzed for cytokine levels by ELISA. Pretreatment with SP-A, Survanta, or SP-A + Survanta significantly decreased the production of TNF-α (Fig. 3A) and IL-6 (Fig. 3B) in response to LPS treatment. These studies suggest that suppression of inflammatory cytokine production is due to the surfactant-mediated upregulation of IRAK-M.

Fig. 3.

SP-A and Survanta pretreatment suppresses LPS-mediated production of proinflammatory cytokines in human macrophages. MDMs were treated with SP-A (10 μg/ml), Survanta (100 μg/ml), or SP-A (10 μg/ml) + Survanta (100 μg/ml) in RPMI containing 1% autologous serum for 12 and 24 h. Cells were washed with warm RPMI and then treated with LPS (100 ng/ml) for 5 h. Cell-free culture supernatants were analyzed for TNF-α (A) and IL-6 (B) production by ELISA. Values represent results from 3 independent experiments (3 donors) performed in triplicate. P < 0.005 for all groups compared with LPS alone (by 1-factor ANOVA). Absolute levels of TNF-α and IL-6 were 2,528 ± 121 and 1,608 ± 95 pg/ml, respectively.

Knockdown of IRAK-M in human macrophages.

To confirm the role of IRAK-M in mediating the effects of surfactant on LPS-generated macrophage cytokine production, we developed an effective and reliable method for knockdown of IRAK-M in MDMs by using IRAK-M-specific siRNA. MDMs were transfected with scramble siRNA or IRAK-M-specific siRNA under optimized conditions and plated in tissue culture plates for 24 h. MDMs were then treated with SP-A, Survanta, or SP-A + Survanta for 12 and 24 h, and cell lysates were analyzed for IRAK-M protein levels by Western blotting. Figure 4, A and B, shows the loss of surfactant-induced expression of IRAK-M protein in MDMs transfected with IRAK-M-specific siRNA at 12 and 24 h. Cell counts of scramble siRNA- and IRAK-M siRNA-transfected MDMs at 24 and 48 h after transfection by naphthol blue black (5) revealed equivalent cell numbers in each experimental group. There was also no difference in monolayer cell viability as determined by Trypan blue exclusion staining (∼95% viable cells in both groups, data not shown).

Fig. 4.

Knockdown of IRAK-M in human macrophages. MDMs were transfected with scramble small interfering RNA (siRNA) or IRAK-M siRNA and incubated in RPMI containing 20% autologous serum for 24 h. Transfected MDMs were treated with SP-A (10 μg/ml), Survanta (Sur, 100 μg/ml), or SP-A (10 μg/ml) + Survanta (100 μg/ml) in RPMI containing 1% autologous serum for 12 h (A) and 24 h (B). Cells were lysed with TN-1 buffer, and IRAK-M protein levels were analyzed by Western blot using an IRAK-M-specific antibody. Western blots are representative of 3 independent experiments.

Knockdown of IRAK-M in surfactant-treated macrophages reverses the IRAK-M-mediated suppression of LPS-induced cytokine production.

The involvement of IRAK-M in TLR4-mediated inflammatory cytokine production was analyzed in IRAK-M-deficient MDMs. IRAK-M siRNA- or scramble siRNA-transfected MDMs were treated with SP-A, Survanta, or SP-A + Survanta for 24 h, washed, and subsequently incubated with LPS for 5 h. Cell-free culture supernatants were analyzed for the production of TNF-α and IL-6 by ELISA. As shown in Fig. 3, control scramble siRNA-transfected surfactant-pretreated macrophages showed a significant reduction in TNF-α and IL-6 production in response to LPS. In contrast, IRAK-M-deficient macrophages showed a complete reversal of this reduction (Fig. 5) compared with LPS alone. Loss of surfactant-induced IRAK-M expression by IRAK-M siRNA was confirmed by Western blotting in each experiment. The results provide evidence that IRAK-M regulates the production of TNF-α and IL-6 in human macrophages in response to the TLR4 ligand LPS.

Fig. 5.

Knockdown of IRAK-M in surfactant-treated macrophages reverses IRAK-M-mediated suppression of LPS-induced cytokine production. MDMs were transfected with scramble siRNA or IRAK-M siRNA and incubated in RPMI containing 20% autologous serum for 24 h. Transfected MDMs were treated with SP-A (10 μg/ml), Survanta (100 μg/ml), or SP-A (10 μg/ml) + Survanta (100 μg/ml) in RPMI containing 1% autologous serum for 24 h and then with LPS (100 ng/ml) for 5 h. Cell-free culture supernatants were analyzed for TNF-α (A) and IL-6 (B) by ELISA. Values represent results from 3 independent experiments performed in triplicate. *P < 0.005, **P < 0.0005.

Pretreatment of human macrophages with SP-A, Survanta, or SP-A + Survanta enhances the production of IL-10 in response to LPS in an IRAK-M-dependent manner.

IL-10 is an immunoregulatory cytokine, and its primary functions are to limit the magnitude and duration of proinflammatory cytokine and chemokine production from macrophages and dendritic cells in response to TLR ligands such as LPS and bacterial lipoproteins (63). LPS induces the production of IL-10 in human AMs (8), and surfactant collectin stimulation enhances the production of IL-10 in mouse AMs (66). We pretreated MDMs with or without SP-A, Survanta, or SP-A + Survanta for 12 and 24 h and subsequently added LPS for 5 h. The cell-free culture supernatants were analyzed for IL-10 cytokine levels by ELISA. We found that surfactant pretreatment enhances the production IL-10 (from 202.4 ± 14.16% to 305.2 ± 18.26%) in response to LPS (Fig. 6A). Thus, in contrast to its effects on proinflammatory cytokine production, surfactant components increase macrophage IL-10 production in response to LPS. Next, we sought to determine whether the LPS-mediated production of IL-10 in surfactant-treated macrophages is due to enhanced IRAK-M expression. IRAK-M siRNA- or scramble siRNA-transfected MDMs were treated with SP-A, Survanta, or SP-A + Survanta for 24 h, washed, and subsequently incubated with LPS for 5 h. The cell-free culture supernatants were analyzed for IL-10 cytokine levels by ELISA. Knockdown of IRAK-M in macrophages nearly abolished IL-10 production in response to LPS. Interestingly, IRAK-M did not affect the low level of LPS-mediated IL-10 production in macrophages not treated with lung surfactant components (Fig. 6B). These results are consistent with the idea that SP-A and Survanta differentiate macrophages toward an M2 phenotype through an IRAK-M-dependent pathway.

Fig. 6.

SP-A and Survanta pretreatment enhances LPS-mediated IL-10 production in human macrophages in an IRAK-M-dependent manner. MDMs were treated with SP-A (10 μg/ml), Survanta (100 μg/ml), or SP-A (10 μg/ml) + Survanta (100 μg/ml) in RPMI containing 1% autologous serum for 12 and 24 h. Cells were washed with warm RPMI and then treated with LPS (100 ng/ml). A: cell-free culture supernatants were analyzed for IL-10 production by ELISA. Results represent cumulative data from 3 independent experiments (3 donors) performed in triplicate. P < 0.005 for all groups compared with LPS alone (by 1-factor ANOVA). B: MDMs were transfected with scramble siRNA or IRAK-M siRNA and incubated in RPMI containing 20% autologous serum for 24 h. Transfected MDMs were treated with SP-A (10 μg/ml), Survanta (100 μg/ml), or SP-A (10 μg/ml) + Survanta (100 μg/ml) in RPMI containing 1% autologous serum for 24 h and then with LPS (100 ng/ml) for 5 h. Cell-free culture supernatants were analyzed for IL-10 production by ELISA. Values (means ± SD) represent results from 2 experiments (2 donors) performed in triplicate. *P < 0.005.

DISCUSSION

The lung alveolar immune microenvironment is unique, in that AMs are influenced by determinants such as the abundantly produced surfactant. Surfactant components are emerging as important determinants in modifying the phenotype and function of resident cells. AMs recognize microbes and inhaled particulates through a subset of PRRs, which effectively clear them with the generation of a highly regulated inflammatory response. SP-A, the most abundantly produced surfactant-associated collectin, interacts with resident AMs to regulate PRRs and their associated immune responses. For example, SP-A enhances the phagocytosis and clearance of opsonized and nonopsonized microbes by regulating the expression of several phagocytic receptors and PRRs, including MR (1), Fcγ receptors, complement receptor 1 (67), scavenger receptor A (38), and TLR2 (26). Less is known about surfactant-associated lipids in these events (10, 22, 50, 73, 74). We previously demonstrated that short-term incubation of SP-A with human macrophages enhances expression of MR and TLR2, but not TLR4 (26). Despite increasing the expression of TLR2, SP-A decreases TLR-mediated TNF-α production by inhibiting NF-κB activation and its associated signaling kinases (26). These events occur quickly (within 30 min) and, thus, mimic more closely those events that would occur upon first contact of macrophages with surfactant. Because AMs are bathed in surfactant, it is also important to examine events in the model over a longer period of time, when regulation that involves transcriptional changes is more likely to occur. Here we show that human AMs express a relatively high level of IRAK-M at baseline compared with MDMs. Thus, although it has been shown that human AMs regulate the immune response in alveoli through increased IL-10 production (4), our current findings provide evidence that IRAK-M in AMs serves as an additional regulator of immune responses in the lung microenvironment (Fig. 7). We show for the first time that expression of IRAK-M increases following longer-term exposure of macrophages to SP-A and/or surfactant lipids (Survanta).

Fig. 7.

Model of SP-A-regulated signaling pathways in human macrophages. Schematic diagram is based on our previous report (26) and current results of how SP-A regulates Toll-like receptor (TLR) signaling through multiple mechanisms: early, by dephosphorylating key biochemical signaling molecules such as Akt and MAPKs and, later, by upregulating negative regulators (i.e., IRAK-M) of the TLR signaling pathway. TRAF6, TNF receptor-associated factor 6.

IRAK-M inhibits TLR-mediated NF-κB activity by binding to MyD88 and TRAF6 (16, 75) and stabilizing the signaling complex (35). Although short-term incubation of macrophages with LPS generates proinflammatory cytokines through TLR4 activation, longer-term incubation leads to increased IRAK-M expression and negative regulation of TLR4-mediated immune responses through the process of LPS tolerance (35, 46). We found that macrophages treated with SP-A and/or Survanta significantly reduce LPS-induced TNF-α and IL-6 production through the enhanced activity of IRAK-M, the latter proven by the use of IRAK-M-deficient human macrophages. In contrast, we found a significant increase in LPS-mediated IL-10 production in human macrophages pretreated with SP-A and/or Survanta. We further demonstrate that IRAK-M is required for LPS-mediated IL-10 production in SP-A- or Survanta-treated macrophages. IL-10, an immunoregulatory and pleiotropic cytokine produced by a variety of immune cells, including macrophages (49), regulates the production of various proinflammatory mediators such as TNF-α, IL-6, IL-8, IL-1β, and IL-12 (14, 19, 23, 58). IL-10 is also a potent inhibitor of antigen-presenting cell function, including dendritic cell maturation (6) and expression of major histocompatibility complex class II and costimulatory molecules (15). IL-10 production is regulated at transcriptional and posttranscriptional levels (57). Studies have shown that IL-10 transcription is governed by several transcriptional factors such as specificity protein 1 (Sp-1) (68), STAT3 (3), and CCAAT/enhancer binding protein (45). The TLR4 ligand LPS induces IL-10 production in human AMs (8) and THP-1 cells (19, 30) through the activation of MAPKs and Sp-1. Our results suggest that surfactant pretreatment may increase the transcriptional activity of Sp-1 and MAPKs. Further studies to address surfactant regulation of the major immune-related transcription activators and the molecular mechanism(s) underlying the involvement of IRAK-M in IL-10 production are underway.

IRAK-M expression has been shown to be regulated by other microbial ligands. For example, M. tuberculosis lipoarabinomannan enhances IRAK-M expression in murine macrophage cell lines (56), LPS from Porphyromonas gingivalis induces IRAK-M expression in THP-1 cells (17), and intranasal administration of influenza in mice upregulates IRAK-M expression in the lungs (62). These results raise the possibility that certain adapted microbes and their determinants can synergize with surfactant components to further upregulate IRAK-M expression, which in turn may enhance their survival following interaction with macrophages. IRAK-M is highly expressed in resting human AMs (34) and tumor-associated macrophages (TAMs) in the lungs compared with peritoneal macrophages (65), indicating the importance of IRAK-M in the lung microenvironment in controlling inflammation. TAMs isolated from the syngeneic mouse model of lung cancer display an M2 phenotype, and TAMs from IRAK-M knockout mice display features of a classically activated M1 phenotype (65). Thus, elevated IRAK-M expression represents another marker of alternatively activated human macrophages.

In the present study, we report that SP-A and/or Survanta induce the expression of IRAK-M in human macrophages. We provide evidence that SP-A- and Survanta-mediated expression of IRAK-M has a direct effect on the production of proinflammatory cytokines following LPS stimulation. Together, these data provide a new mechanism whereby surfactant components regulate the innate immune response of human macrophages in the lung.

GRANTS

This work was supported by National Institute on Aging Grant AI-059639 (L. S. Schlesinger).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

H.A.N., M.V.S.R., and D.A.M. performed the experiments; H.A.N., M.V.S.R., D.A.M., and L.S.S. analyzed the data; H.A.N. and M.V.S.R. prepared the figures; H.A.N. and M.V.S.R. drafted the manuscript; M.V.S.R. and L.S.S. are responsible for conception and design of the research; M.V.S.R. and L.S.S. interpreted the results of the experiments; M.V.S.R. and L.S.S. edited and revised the manuscript; L.S.S. approved the final version of the manuscript.