Interleukin 6 mediates the therapeutic effects of adipose-derived stromal/stem cells in lipopolysaccharide-induced acute lung injury

Abstract

Adipose-derived stromal/stem cells (ASCs) have anti-inflammatory as well as immunosuppressive activities and are currently the focus of clinical trials for a number of inflammatory diseases. Acute lung injury (ALI) is an inflammatory condition of the lung for which standard treatment is mainly supportive due to lack of effective therapies. Our recent studies have demonstrated the ability of both human ASCs (hASCs) and mouse ASCs (mASCs) to attenuate lung damage and inflammation in a rodent model of lipopolysaccharide-induced ALI, suggesting that ASCs may also be beneficial in treating ALI. To better understand how ASCs may act in ALI and to elucidate the mechanism(s) involved in ASC modulation of lung inflammation, gene expression analysis was performed in ASC-treated (hASCs or mASCs) and control sham-treated lungs. The results revealed a dramatic difference between the expression of anti-inflammatory molecules by hASCs and mASCs. These data show that the beneficial effects of hASCs and mASCs in ALI may result from the production of different paracrine factors. Interleukin 6 (IL-6) expression in the mASC-treated lungs was significantly elevated as compared to sham-treated controls 20 hours after delivery of the cells by oropharyngeal aspiration. Knockdown of IL-6 expression in mASCs by RNA interference abrogated most of their therapeutic effects, suggesting that the anti-inflammatory properties of mASCs in ALI are explained, at least in part, by activation of IL-6 secretion.

Keywords: Acute lung injury; Adipose-derived stromal/stem cell; Interleukin 6; Mouse.

Figure 1

Lung histology and expression of anti-inflammatory/trophic factors of human origin in the lungs of acute lung injury (ALI) mice treated with hASCs. Both hASC and mASC treatments improved lung injury as assessed by histological methods. (A): H&E staining of lung sections demonstrated attenuated lung injury in the mice treated with hASCs and mASCs at 72 hours after ALI induction. (B): Quantification of lung injury showed a significant reduction in the degree of inflammation and hemorrhage in the mice receiving hASCs and mASCs compared to mice receiving HBSS. N = 4 for each group. Significance was defined as * and ** for p < .05 and p < .01, respectively, compared to HBSS sham treatment. Steady-state mRNA levels of (C) hTFPI-2, (D) hTGF-β, (E) hSTC-1, (F) hANGPT-1, (G) hKGF, (H) hIL1RN, (I) hTSG-6, (J) hLIF, (K) hiNOS, (L) hIL-6, and (M) hIDO are shown for PBS- or LPS- challenged mice injected with hASCs. All levels were normalized to GAPDH and reported as fold changes compared to in vitro control. N = 4 for both PBS1hASC and LPS1hASC groups. Significance was defined as *, **, and *** for p < .05, p < .01, and p < .001, respectively, compared to in vitro control; significant differences between PBS1hASC and LPS1hASC groups were denoted by #, ##, and ### for p < .05, p < .01, and p < .001, respectively. Abbreviations: hANGPT-1, human angiopoietin 1; HBSS, Hank's balanced salt solution; hASC, human adipose-derived stem cell; hIDO, human indoleamine 2,3-dioxygenase; hIL-6, human interleukin; hIL1RN, human interleukin 1 receptor antagonist; hLIF, human leukemia inhibitory factor; hKGF, human keratinocyte growth factor; hTFPI-2, human tissue factor pathway inhibitor 2; hTGF-β, human transforming growth factor beta; hTSG-6, human tumor necrosis factor alpha-induced protein 6; hSTC-1, human stanniocalcin 1; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; mASC, mouse adipose-derived stem cells; PBS, phosphate buffered saline.

Figure 2

Expression of anti-inflammatory/trophic factors of mouse origin in the lungs of acute lung injury (ALI) mice treated with hASCs and mASCs. Steady-state mRNA levels of (A) mTFPI-2, (B) mTGF-β, (C) mSTC-1, (D) mANGPT-1, (E) mKGF, (F) mIL1RN, (G) mTSG-6, (H) mLIF, (I) miNOS, and (J) mIL-6 are shown for PBS- or LPS-challenged mice injected with HBSS, hASCs, or mASCs. All levels were normalized to beta actin and reported as fold changes compared to unchallenged and untreated controls (Intact). N = 2, 4, 4, 5, 4, and 5 for Intact, PBS+hASC, PBS+mASC, LPS+HBSS, LPS+hASC, and LPS+mASC, respectively. Significance was defined as *, **, and *** for p < .05, p < .01, and p < .001, respectively, compared to control groups; significant differences between the LPS-challenged cell-treatment groups (LPS+hASC and LPS+mASC) were denoted by #, ##, and ### for p < .05, p < .01, and p < .001, respectively. Abbreviations: hASC, human adipose-derived stem cells; HBSS, Hanks’ balanced salt solution; LPS, lipopolysaccharide; mASCs, mouse adipose-derived stem cells; mTFPI, mouse tissue factor pathway inhibitor 2; mTGF-β, mouse transforming growth factor beta; mSTC-1, mouse stanniocalcin 1; mANGPT-1, mouse angiopoietin 1; mKGF, mouse keratinocyte growth factor; mILRN, mouse interleukin 1 receptor antagonist; mTSG-6, mouse tumor necrosis factor, alpha-induced protein 6; mILF, mouse leukemia inhibitory factor; miNOS, mouse inducible nitric oxide synthase; mIL-6, mouse interleukin 6; PBS, phosphate buffered saline.

Figure 3

LIF and IL-6 in BALF and lungs from mASC- and hASC-treated acute lung injury (ALI) mice. Protein levels of LIF and IL-6 in BALF (A, C) and lung lysates (B, D) were quantified using a mouse Bio-Plex immunoassay 24 hours after PBS or LPS challenge. Values for LIF and IL-6 in lung lysates were normalized to total lung lysate protein. N = 4, 4, 5, 4, and 5 for PBS+hASC, PBS+mASC, LPS+HBSS, LPS+hASC, and LPS+mASC, respectively. Significance was defined as *** for p < .001 compared to LPS+HBSS; significant differences between LPS+hASC and LPS+mASC groups were denoted by ### for p < .001. Abbreviations: BALF, bronchoalveolar lavage fluid; HBSS, Hanks’ balanced salt solution; hASCs, human adipose-derived stem cells; LPS, lipopolysaccharide; PBS, phosphate buffered saline; mIL-6, mouse interleukin-6; mLIF, mouse leukemia inhibitory factor; mASCs, mouse adipose-derived stem cells.

Figure 4

Assessing gene expression by mouse adipose-derived stem cells (mASCs) upon stimulation with BALF. The expression of various genes by mASCs was quantified using real-time reverse transcription polymerase chain reaction. Steady-state mRNA levels of (A) mTFPI-2, (B) mTGF-β, (C) mSTC-1, (D) mANGPT-1, (E) mKGF, (F) mIL1RN, (G) mTSG-6, (H) mLIF, (I) miNOS, and (J) mIL-6 are shown for mASCs stimulated by BALF or lavage buffer alone at 6 hours and 20 hours poststimulation. All expression levels were normalized to beta actin expression and reported as fold changes compared to lavage buffer stimulated controls (Ctrl). N = 6 for each group. Significance was defined as *** for p < .001, compared to control groups. Abbreviations: BALF, bronchoalveolar lavage fluid; mTFPI, mouse tissue factor pathway inhibitor 2; mTGF-b, mouse transforming growth factor beta; mSTC-1, mouse stanniocalcin 1; mANGPT-1, mouse angiopoietin 1; mKGF, mouse keratinocyte growth factor; mILRN, mouse interleukin 1 receptor antagonist; mTSG-6, mouse tumor necrosis factor, alpha-induced protein 6; mILF, mouse leukemia inhibitory factor; miNOS, mouse inducible nitric oxide synthase; mIL-6, mouse interleukin 6.

Figure 5

Evaluation of IL-6 knockdown efficiency in mASCs. The IL-6 knockdown efficiency in mASCs was quantified using real-time reverse transcriptase polymerase chain reaction (RT-PCR) and ELISA. (A): Steady-state mRNA levels of IL-6 are shown for mASCs with or without siRNA transfection followed by LPS stimulation for 20 hours. All expression levels were normalized to beta actin expression and reported as fold changes compared to mASCs without LPS activation (Control). N = 3 for each group. Significance was defined as *** for p < .001, compared to control group. (B): The in vivo lung mRNA levels of IL-6 24 hours after LPS challenge were quantified using real-time RT-PCR. Steady-state mRNA levels of IL-6 are shown for LPS-challenged mice injected with HBSS, mASCs transfected with Silencer Select Negative Control No. 1 (NC) siRNA, or mASCs transfected with IL-6 siRNA. All expression levels were normalized to beta actin expression and reported as fold changes compared to unchallenged and untreated controls (Intact). N = 2, 6, 5, and 4 for Intact, LPS+HBSS, LPS+mASC/NC siRNA, and LPS+mASC/IL-6 siRNA, respectively. The protein levels of IL-6 in BALF (C) and lung lysates (D) 24 hours after LPS challenge were quantified by ELISA. IL-6 levels are shown for intact mice and LPS-challenged mice injected with HBSS, mASCs transfected with NC siRNA, or mASCs transfected with IL-6 siRNA. Values for IL-6 in lung lysates were normalized to total lung lysate protein. N = 3, 6, 5, and 4 for Intact, LPS+HBSS, LPS+mASC/NC siRNA, and LPS+mASC/IL-6 siRNA, respectively. Significance was defined as *** for p < .001, compared to LPS1HBSS group; significant differences between LPS+mASC/NC siRNA and LPS+mASC/IL-6 siRNA groups were denoted by ### for p < .001. Abbreviations: BALF, bronchoalveolar lavage fluid; HBSS, Hanks’ balanced salt solution; mASCs, mouse adipose-derived stem cells; LPS, lipopolysaccharide; NC, negative control; mIL-6, mouse interleukin-6; siRNA, small interfering RNA.

Figure 6

Effects of IL-6 knockdown mASCs on BALF protein and albumin levels, inflammatory cell infiltration, and MPO activity in the lungs after LPS injury. Levels of total protein (A) and albumin (B) in the BAL fluid were used to assess the extent of vascular leakage after treatment with mASCs transfected with either NC siRNA or IL-6 siRNA. Intact or HBSS-treated mice were used as controls. Values were presented as fold changes relative to unchallenged and untreated controls (Intact). Administration of NC siRNA-transfected mASCs, but not IL-6 siRNA-transfected mASCs, significantly decreased the (C) total infiltrating cells, (D) neutrophils, and (E) MPO activity in BALF 24 hours after LPS exposure when compared to HBSS sham treatment controls. N = 3, 6, 5, and 4 for intact, LPS+HBSS, LPS+mASC/NC siRNA, and LPS+mASC/IL-6 siRNA groups, respectively. Significance was defined as ** and *** for p < .01 and p < .001, respectively, as compared to the HBSS-treated LPS-challenged mice; significant differences between LPS+mASC/NC siRNA and LPS+mASC/IL-6 siRNA groups were denoted by # for p < .05. Abbreviations: BAL, bronchoalveolar lavage; HBSS, Hanks’ balanced salt solution; IL-6, interleukin-6; LPS, lipopolysaccharide; MPO, myeloperoxidase; mASCs, mouse adipose-derived stem cells; NC, negative control; siRNA, small interfering RNA.

Figure 7

Effects of IL-6 knockdown mASCs on inflammatory responses in LPS-treated lung. The lung mRNA levels of various cytokines and chemokines produced in response to LPS challenge were quantified 24 hours after exposure using real-time reverse transcriptase polymerase chain reaction. Steady-state mRNA levels of (A) MIP-2, (B) IL-1α, (C) IL-1β, (D) MIP-1α, (E) TNF-α, and (F) IL-10 are shown for each group. All expression levels were normalized to beta actin expression and reported as fold changes compared to unchallenged and untreated mouse levels. N 5 3, 6, 5, and 4 for Intact, LPS1HBSS, LPS+mASC/NC siRNA, and LPS+mASC/IL-6 siRNA, respectively. Significance was defined as * and *** for p < .05 and p < .001, respectively, as compared to the HBSS-treated LPS-challenged mice; signifi-cant differences between LPS1mASC/NC siRNA and LPS+mASC/IL-6 siRNA groups were denoted by # and ## for p < .05 and p < .01, respectively. Abbreviations: HBSS, Hanks’ balanced salt solution; IL-6, interleukin-6; LPS, lipopolysaccharide; mSACs, mouse adipose-derived stem cells; mMIP-2, mouse macrophage inflammatory protein-2; mIL-1α, mouse interleukin 1α; mTNF-α, mouse tumour necrosis factor a; siRNA, small interfering RNA.