Role of hypoxia-inducible factor 1{alpha} in modulating cobalt-induced lung inflammation

Abstract

Hypoxia plays an important role in development, cellular homeostasis, and pathological conditions, such as cancer and stroke. There is also growing evidence that hypoxia is an important modulator of the inflammatory process. Hypoxia-inducible factors (HIFs) are a family of proteins that regulate the cellular response to oxygen deficit, and loss of HIFs impairs inflammatory cell function. There is little known, however, about the role of epithelial-derived HIF signaling in modulating inflammation. Cobalt is capable of eliciting an allergic response and promoting HIF signaling. To characterize the inflammatory function of epithelial-derived HIF in response to inhaled cobalt, a conditional lung-specific HIF1alpha, the most ubiquitously expressed HIF, deletion mouse, was created. Control mice showed classic signs of metal-induced injury following cobalt exposure, including fibrosis and neutrophil infiltration. In contrast, HIF1alpha-deficient mice displayed a Th2 response that resembled asthma, including increased eosinophilic infiltration, mucus cell metaplasia, and chitinase-like protein expression. The results suggest that epithelial-derived HIF signaling has a critical role in establishing a tissue's inflammatory response, and compromised HIF1alpha signaling biases the tissue towards a Th2-mediated reaction.

Fig. 1.

Hypoxia-inducible factor 1α (HIF1α) immunohistochemistry of lungs from control and doxycycline-treated mice. Lung tissue sections from control (A and B) and mice that were HIF1α deficient in their lungs (HIF1αΔ/Δ) (C and D) were analyzed by immunohistochemistry using a HIF1α-specific antibody. Control (A and B) and doxycycline (C and D)-treated mice were compared. HIF1α staining is prominent in the epithelial cell (e) lining the bronchiolar airway (BA) (dashed arrow) and type II cells (solid arrow) in the alveolar duct (AD) and alveolus (a). Staining is greatly reduced in the postnatally doxycycline-treated animals (C and D).

Fig. 2.

Weight change and cell counts from cobalt-challenged control and HIF1αΔ/Δ mice. Mice were exposed to saline and 30 μg (5 mM) or 60 μg (10 mM) of CoCl₂ for 2 wk, 5 days/wk paradigm. Total animal weight change (A) in control (white bars) and HIF1αΔ/Δ (black bars) was calculated by subtracting animal weight on the day of death from weight at the start of exposure. Total cell counts from BALF (B) were assessed from all mice and averaged. Cell differential counts were performed for macrophages (C), lymphocytes (D), neutrophils (E), and eosinophils (F). N > 5 mice/group. *Significance (P values noted).

Fig. 3.

Histopathology and picrosirius staining control and cobalt-treated control mice. Light photomicrographs of the lungs of control mice instilled with saline (A and C) or cobalt (B and D). Lung sections in A and B were stained with hematoxylin and eosin, whereas sections in C and D were histochemically treated with picrosirius red solution that stains interstitial collagen (red chromagen in interstitial tissues in the alveolar septa and around bronchiolar airways). In cobalt-treated lungs (B and D), there is marked inflammation and interstitial fibrosis (* in B) around preterminal bronchioles (ptb) and terminal bronchioles (tb) and extending distally into alveolar ducts (ad) and adjacent alveoli (a). There is increased picrosirius red-stained collagen (solid arrows) in the alveolar septa of the cobalt-treated mouse (D) compared with that in the saline-treated control mouse (C). p, Pulmonary pleura; e, bronchiolar epithelium; dashed arrow, mixed inflammatory cell infiltrate.

Fig. 4.

Major basic protein staining in lungs from control and HIF1αΔ/Δ mice. Light photomicrographs of the lungs of saline- (A and C) or cobalt-instilled (B and D) control (A and B) and HIF1αΔ/Δ (C and D) mice. All lung sections were immunohistochemically stained for major basic protein to identify infiltrating eosinophils (red chromagen; arrows) and counterstained with hematoxylin. A mixed inflammatory infiltrate consisting of mononuclear leukocytes, eosinophils, and lesser numbers of neutrophils are restricted to the lungs in cobalt-treated mice (B and D). Markedly more eosinophils are present in the peribronchiolar and alveolar regions of the cobalt-treated HIF1αΔ/Δ mouse (D) compared with that of the cobalt-treated control mouse (B). pa, Pulmonary arteriole; a, alveolar air space; e, bronchiolar epithelium.

Fig. 5.

Alcian Blue/periodic acid-Schiff (AB/PAS) stain and YM1/2 immunohistochemistry (IHC). Light photomicrographs of ptb of cobalt-instilled control (A and C) and HIF1αΔ/Δ (B and D) mice. Tissues were stained with AB (pH 2.5)/PAS (A and B) to identify acidic and neutral mucosubstances (magenta stain; arrows) in mucous cells within the bronchiolar epithelium (e). Numerous AB/PAS-stained mucous cells are present only in the airway epithelium lining the preterminal bronchiole in the cobalt-treated HIF1αΔ/Δ mouse (B). Tissues in C and D were immunohistochemically stained for YM1/2 protein (brown chromagen; arrows) and counterstained with hematoxylin. YM1/2 proteins were present only in the bronchiolar epithelium of the cobalt-treated HIF1αΔ/Δ mouse (D). Arrow in C identifies a few alveolar macrophages that were positive for YM1/2 proteins.

Fig. 6.

Hematoxylin and eosin (H&E) staining and YM1/2 IHC of cobalt-treated control and HIF1αΔ/Δ mice. Light photomicrographs of alveolar macrophages in cobalt-instilled control (A and C) and HIF1αΔ/Δ (B and D) mice. Lung tissues were histochemically stained with H&E (A and B) or immunohistochemically for YM1/2 proteins (C and D). H&E-stained alveolar macrophages in the cobalt-instilled HIF1αΔ/Δ mouse (B) are larger (hypertrophic) and more eosinophilic than the similarly stained lung section from the cobalt-instilled control mouse (A). In B, a group of macrophages is surrounding an extracellular aggregate of eosinophilic crystals (dashed arrow). In addition, more alveolar macrophages immunohistochemically staining for YM1/2 proteins (solid arrows) are present in lung section from the cobalt-instilled HIF1αΔ/Δ mouse (D) compared with that of the cobalt-instilled control mouse (C). Dashed arrows in C, alveolar macrophage with no detectable YM1/2 proteins.

Fig. 7.

Gene expression results. The expression of 63 genes was analyzed by qRT-PCR. Those genes that showed a difference as pooled samples were further analyzed as independent replicates. Expression levels were normalized to the saline-treated control animal and expressed as fold change. ^aP < 0.07, *P < 0.05, **P < 0.01.