TLR4 is a negative regulator in noninfectious lung inflammation

Abstract

Low m.w. hyaluronan (LMW HA) has been shown to elicit the expression of proinflammatory cytokines and chemokines in various cells in vitro. However, the effects of this molecule in vivo are unknown. In this study, we report that intratracheal administration of LMW HA (200 kDa) causes inflammation in mouse lung. A lack of TLR4 is associated with even stronger inflammatory response in the lung as shown by increased neutrophil counts and elevated cytokine and chemokine concentrations. We also demonstrate that TLR4 anti-inflammatory signaling is dependent upon a MyD88-independent pathway. TLR4-mediated IL-1R antagonist production plays a negative regulatory role in LMW HA (200 kDa) induced lung inflammation. These data provide a molecular level explanation for the function of TLR4 in LMW HA (200 kDa)-induced lung inflammation, as inhibition of the beta form of pro-IL-1 promotes an anti-inflammatory response.

FIGURE 1.

TLR4 deficiency is associated with exacerbated LMW HA-induced lung injury. A, The elution profile of a LMW HA (200 kDa), prepared by gamma-irradiation of bacterially fermented HA, using SEC/TriSEC 302 detectors. The eluted peaks were Gaussian and did not exhibit tailing, shoulders, or extraneous peaks (red, refractive index; green, right-angle light scattering; black, low-angle light scattering; blue, differential pressure viscometer). B, Twenty-four hours after intratracheal administration of LMW HA (200 kDa, 65 mg/kg), pathological observation of the lung. C, Lung sections were stained with H&E, original magnification ×200. D, Diff-Quick–stained cytospins of bronchoalveolar lavage neutrophils, original magnification ×400. The BALF dilution in TLR4^−/− plus LMW HA of D was 1/4. Arrows indicate RBCs. E, Lung injury score. F, Total cell and neutrophil counts were performed on BALF. G, After BALF was performed, the MPO of whole lung homogenates was measured. H, BALF neutrophil influx in two strains of TLR4-deficient/mutant mice (C57BL/6J, C3H/HeJ) and respective controls (C57BL/6J, C3H/HeOuJ). After 1, 3, and 7 d following LMW HA, neutrophils in BALFs were quantified (I), and MPO in lung tissues was analyzed (J). B–D, Representative data from multiple mice (n = 4–6 per group) are shown. Data represent mean ± SEM from three independent experiments (n = 4 mice per group). *p < 0.05.

FIGURE 2.

LMW HA (200 kDa) is the only source to drive the lung inflammation in 24 h after LMW HA. LMW HA alone or postdigestion with hyaluronidase (HAase), pronase or DNase I (DNAse) were administered into the tracheas of TLR4^−/− mice. BALF neutrophils were measured. Blockade of LMW HA (200 kDa) by Pep-1 or hyaluronidase (HAase) ameliorated lung injury in TLR4^−/− mice. Data represent mean ± SEM from two independent experiments (n = 4 mice per group). *p < 0.05.

FIGURE 3.

TLR4 deficiency is associated with increased cytokine/chemokine responses and hyperpermeability in the lung 24 h after intratracheal administration of LMW HA (200 kDa, 65 mg/kg). A, IL-1β. B, TNF-α. C, IL-6. D, MIP-2. E, KC. Albumin (F) and IgM (G) were upregulated in the BALF of TLR4^−/− mice. Data represent mean ± SEM from three independent experiments (n = 4 mice per group). *p < 0.05.

FIGURE 4.

MyD88-independent pathway plays a protective role in LMW HA-induced lung injury 24 h after intratracheal administration of LMW HA (200 kDa, 65 mg/kg). A, Increased neutrophil infiltration and bleeding in TLR4^−/−, TRAM–TRIF double^−/−, and IRF3^−/− but not MyD88^−/− and TRIF^−/− mice (H&E, original magnification ×400). Representative results of multiple mice (n = 5–8 per group) are shown. Lung injury scores (B), BALF neutrophils (C), IL-1β (D), and IL-1RA (E) in WT, TLR4^−/−, MyD88^−/−, TRAM–TRIF double^−/−, and IRF3^−/− mice. All mice were on the C57BL/6J background. Data represent mean ± SEM from three independent experiments (n = 4 mice per group). *p < 0.05.

FIGURE 5.

Increased LMW HA-induced inflammatory responses are associated with the imbalance between IL-1b and IL-1RA in TLR4^−/− mice. A, Neutrophils. B, IL-1β. C, MIP-2. D, KC. TNF-α (E) and IL-6 (F) BALF levels were analyzed 24 h after intratracheal administration of LMW HA in the presence of IL-1RA (A–F) or TNF-α inhibitor (A–D). The amount of IL-1RA in BALFs (G) and IL-1RA/IL-1β ratio (H) were decreased in TLR4^−/− mice 24 h after LMW HA. BALF neutrophils that had been isolated from mice for 24 h after intratracheal administration of LMW HA were restimulated with LMW HA in vitro for 12 h. Then the production of IL-1RA (I) and IL-1β (J) was measured. K, IL-1RA/IL-1β ratio in neutrophils. Data represent one of three independent experiments with similar results (n = 4 mice per group). *p < 0.05.