Deletion of the lactoperoxidase gene causes multisystem inflammation and tumors in mice

Abstract

Strongly oxidative H₂O₂ is biologically important, but if uncontrolled, would lead to tissue injuries. Lactoperoxidase (LPO) catalyzes the redox reaction of reducing highly reactive H₂O₂ to H₂O while oxidizing thiocyanate (SCN^-) to relatively tissue-innocuous hypothiocyanite (OSCN^-). SCN^- is the only known natural, effective reducing-substrate of LPO; humans normally derive SCN^- solely from food. While its enzymatic mechanism is understood, the actual biological role of the LPO-SCN^- system in mammals remains unestablished. Our group previously showed that this system protected cultured human cells from H₂O₂-caused injuries, a basis for the hypothesis that general deficiency of such an antioxidative mechanism would lead to multisystem inflammation and tumors. To test this hypothesis, we globally deleted the Lpo gene in mice. The mutant mice exhibited inflammation and lesions in the cardiovascular, respiratory, digestive or excretory systems, neuropathology, and tumors, with high incidence. Thus, this understudied LPO-SCN^- system is an essential protective mechanism in vivo.

Figure 1

Reaction schemes, survival curves, body weight and incidents of histological findings in major organs of Lpo^del cohort. (a) LPO-catalyzed oxidation reaction of SCN⁻ by H₂O₂ to OSCN⁻. (b) Two competing MPO-catalyzed reactions: oxidation of SCN⁻ to OSCN⁻ and Cl⁻ to OCl⁻. (c) Percentage of wild-type (black line) and mutant (magenta curve) mice remaining at one year of age. All 16 male and 23 female wild-type mice remained whereas 28 out of 45 male and 40 out 53 female mutant mice remained. Nine mutant mice found dead and 21 euthanized for humane endpoints. (d) Dot plots of body weights of individual one-year-old wild-type (black) and mutant (magenta) mice, where the mean and s.e.m. are presented to the right of the respective groups. The mean (± s.e.m.) body weight in grams was 41.2 (± 2.00) for male mutant mice (magenta diamonds, n = 19), compared with 34.7 (± 1.02) for male wild-type mice (black diamonds, n = 10), or 32.2 (± 1.33) for female mutant mice (magenta circles, n = 30), compared with 27.9 (± 0.92) for female wild-type mice (black circles, n = 15). P value by two-tailed Welch’s t test is 7.24 × 10^–3 for the male or 1.10 × 10^–2 for the female group. (e) Examples of a male wild-type mouse of typical body weight and an obese mutant mouse. (f,g) Illustration of the excessive body fat of an obese mutant mouse (f), which weighed 10 g on a scale (g). (h) Summary of the incidence of notable histological findings in major types of organs of Lpo^del mice, compared with that of the wild type. The numbers in individual boxes indicate the count of male or female wild-type (black boxes) or mutant (magenta boxes) mice with the “(−)" or "(+)" designation for individual organ types.

Figure 2

Cardiac histopathology of Lpo^del mice. (a) Digitized scans of MT-stained sections of hearts from two wild-type (left) and two mutant (right) mice. In the mutant sample, left and right ventricles (lv and rv) were dilated, with thinner ventricular walls (arrowheads). (b–h) Histomicrographs of cardiac tissue sections of wild-type (left) or mutant (right) mice, stained by HE unless specified otherwise. (b) Sections of part of the left ventricle. The mutant sample exhibited inflammatory infiltrates (arrow) underneath the endocardium and myocytes degeneration (closed arrowhead) with apparent fibrosis (open arrowhead). (c) Sections of the coronary artery. In the mutant sample, infiltrating leukocytes (arrow) and apparent fibrosis (arrowhead) were present around a coronary artery, which contained an apparent greyish plaque (asterisk). (d) MT-stained sections where the mutant sample exhibited infiltrating leukocytes (arrow) and heavy collagen deposition (blue) around the narrowed coronary artery (asterisk). (e,f) Sections of the aorta exhibiting clusters of infiltrating leukocytes (arrows) in the mutant samples. MT-stained cross-sections revealed increased collagen deposition (blue, f) in and around the aortic wall of the mutant sample. (g) MT-stained sections displaying the tricuspid valves pointed at by the arrowheads. The mutant sample exhibited infiltrating leukocytes (arrow) and increased collagen deposition (blue). (h) Sections displaying the mitral valves pointed at by the arrowheads. The mutant sample exhibited infiltrating leukocytes (arrow) with apparent fibrosis in the mitral valve. Scale bars are 25 µm (c–e); 50 µm (b); 100 µm (g); 250 µm (f,h); 2 mm (a).

Figure 3

Pulmonary histopathology of Lpo^del mice. (a–d) HE-stained sections. (a) The wild-type tissue section (left) exhibited a single small BALT (arrow), whereas the mutant section (right) exhibited numerous larger clusters around bronchioles (br, black arrows) and vasculature (va, blue arrows), with only some clusters indicated by arrows. (b) The mutant sample displayed infiltrating leukocytes around bronchioles (black arrow) or vasculature (blue arrow), and histiocytes (yellow arrows) in alveoli. (c,d) The mutant samples showed infiltrating leukocytes (black arrows, c) scattered among diffuse, hyperplastic type II pneumocytes (arrowheads, c) in a region with loss of alveolar structure (cyan asterisks, c) or clustered infiltrating leukocytes (blue arrows, d) in a thickened alveolar septum with type II pneumocyte hyperplasia (arrowhead, d). (e) MT-stained sections exhibiting bronchioles. The mutant sample exhibited hypercellular epithelium (arrowheads), thickened muscularis (yellow asterisks), a patch of leukocyte infiltrates (arrow), and heavier collagen deposition (blue). (f) Sections containing an intrapulmonary bronchial branch where mucin in the epithelium and glycoproteins in the basement membranes were stained purple by ABPASH. In the mutant sample, mucin-producing goblet cells (arrowheads) in the epithelium were hyperplastic, and the basement membranes were thickened. There were a large patch of peribronchial inflammatory infiltrates (arrow) and a piece of mucus in the lumen (asterisk). Scale bars are 25 µm (c,d); 50 µm (e); 100 µm (b,f); 500 µm (a).

Figure 4

Renal histopathology of Lpo^del mice. (a–f) Tissue samples showing areas of the renal cortex of wild-type (left) or mutant (right) mice where only samples in (a) contained also part of medulla. Sections were stained by HE unless specified otherwise. (a,b) Sections where the mutant sample exhibited infiltrating leukocytes (arrows), and eosinophilic proteinaceous material stained pink (asterisks, b) in or around pathological glomeruli and in adjacent renal tubules. (c) PASH-stained sections where the mutant sample exhibited increased glycoproteins (stained purple) in the GBM and the adjacent tubule wall (arrowheads), accompanied by some infiltrating leukocytes (arrows) in the tubulointerstitium. (d,e) MT-stained sections where the mutant samples revealed: (i) increased collagen deposition (blue) in or around the pathological glomeruli, or around tubules and the vasculature; (ii) infiltrating mononuclear (black arrows) and scattered polymorphonuclear (yellow arrow, e) leukocytes in the perivascular space and the tubulointerstitium around the affected glomeruli. (f) Sections where the mutant sample exhibited an atrophic glomerulus with enlarged Bowman’s space (asterisk) and a cluster of infiltrating leukocytes (arrows) between a glomerulus and an artery. Scale bars are 25 µm (d); 50 µm (b,c,e,f); 250 µm (a).

Figure 5

Histopathology in the small intestines and colon of Lpo^del mice. (a–e) Longitudinal sections of indicated regions of wild-type (left) or mutant (right) small intestines, stained by HE unless specified. (a) Sections showing Paneth cells containing eosinophilic granules (blue arrowheads) or goblet cells containing unstained mucin granules (magenta arrowheads). The mutant sample displayed hyperplastic Paneth and goblet cells plus thickened muscularis (asterisk). (b,c) The mutant samples exhibited hypertrophic Paneth cells (blue arrowheads, b), or infiltration of mononuclear (black arrow) and polymorphonuclear leukocytes (yellow arrow, c) between villi (asterisks). Sections in panel (c) were perpendicular to villi. (d) ABPASH stains revealing increased mucin (blue or red, together giving purple) in the goblet cells (magenta arrowheads) or secreted mucin (yellow arrowhead) between villi in the mutant sample. (e) MT stain revealing collagen deposition (blue). The mutant sample displayed hypercellular (black arrowheads) and hyperplastic (cyan arrowhead) villi, and thickened muscularis (asterisk). (f–j) Longitudinal sections of indicated regions of the wild-type (left) or mutant (right) colon, stained by HE unless specified. (f) Sections exhibiting one small GALT (arrow) in the submucosa of the wild-type sample and multiple larger leukocyte clusters (arrows) in the mutant sample. (g,h) Sections where the mutant sample exhibited, in the mucosa or submucosa, mononuclear (black arrow, g) and polymorphonuclear leukocytes (yellow arrow, g), apparent fibrosis (lime arrowheads, g), erosion of mucosa (open arrowhead, h), and cellular sloughing (closed arrowheads, h). (i) ABPASH-stained sections where the mutant sample exhibited hyperplastic goblet cells (magenta arrowheads) and crypt units (cyan arrowheads), excessive secreted mucin (yellow arrowhead), and leukocyte infiltrates (black arrow) in the mucosa. (j) MT-stained sections revealing collagen deposition (blue), with mononuclear (black arrow) and polymorphonuclear leukocytes (yellow arrow) in the submucosa of mutant sample. Scale bars are 25 µm (b,c,g,j); 50 µm (i); 100 µm (a,d,e,h); 500 µm (f).

Figure 6

Histopathology in the liver, gallbladder, cystic duct and pancreas of Lpo^del mice. All tissue sections of wild-type (left) or mutant (right) mice were stained by HE unless specified otherwise. (a,b) The mutant liver samples exhibited: (i) mononuclear (black arrow), polymorphonuclear (yellow arrow) inflammatory infiltrates and degenerative hepatocytes (black arrowheads) in the parenchyma (a), or primarily mononuclear infiltrates in the hepatic vascular wall (black arrows, b); (ii) increased collagen deposition in and around the vascular wall (blue, b). (c) ABPASH-stained sections of the gallbladder. In the mutant sample, accumulated acidic mucosubstance (black arrowheads) in the gallbladder epithelium was stained dark blue. (d) Sections of the cystic duct where the mutant sample displayed diffusive inflammatory infiltrates (black arrow) in the wall and hyaline droplets and crystals (yellow arrowheads) in the epithelium of the cystic duct. (e–h) Pancreas sections. (e) Sections of the pancreas containing an intralobular duct, around which were a few mononuclear leukocytes (cyan arrow, left) in the wild-type sample but numerous inflammatory infiltrates (cyan arrows, right) in the mutant sample. (f) MT-stained sections of the pancreas revealing deposition of collagen (blue). There were some mononuclear leukocytes (black arrow, left) around an interlobular duct in the wild-type sample, but markedly more (black arrow, right) in the mutant sample with scattered polymorphonuclear leukocytes (yellow arrow, right). (g) Sections containing pancreatic islets of Langerhans where the islet in the mutant sample was infiltrated by numerous mononuclear (black arrow) and polymorphonuclear (yellow arrow) leukocytes. (h) Sections of the pancreas where the mutant sample exhibited a patch of swollen acinar cells with hypereosinophilic cytoplasm and apparent pyknotic nucleus (yellow arrowheads). Scale bars are 25 µm (a,g); 50 µm (e,f,h); 100 µm (b,d); 250 µm (c).

Figure 7

Pathology in the brain of Lpo^del mice. (a) Coronal slices of fixed brains at levels 2 or 3^, from wild-type (left) and mutant (right) mice. The lateral ventricles (LV) in the mutant sample were dilated. (b–d) HE-stained tissue sections of the brain from wild-type (left) or mutant (right) mice. (b) The mutant sample exhibited enlarged LV. (c) Sections around part of LV where the mutant sample exhibited enlarged LV and irregular choroid plexus with hyperplastic epithelium (arrowheads). (d) Sections around the third ventricle (TV) where the mutant sample exhibited flattened ependymal cells (arrowheads). The adjacent neuropil had a foamy appearance (asterisk). Scale bars are 25 µm (d); 100 µm (c); 500 µm (b); 2 mm (a).

Figure 8

Multiple types of tumors observed in various organs of Lpo^del mice. (a–l) HE-stained sections of tissue samples from mutant mice. (a) Section of a lung tissue exhibiting an apparent carcinoma. (b–h) Sections exhibiting a lymphoma adjacent or attached to the following organs or tissues: heart (b), mesentery (c), pancreas (d), salivary glands (e), lung (f); lymphoma in the spleen (g), or in the mucosa of the jejunum (h). (i) Section of a skin tissue sample exhibiting a pleomorphic sarcoma with giant cells. (j–l) Sections exhibiting a histiocytic sarcoma in the spleen (j), near a portal vein in the liver (arrowhead, k) or in the bone marrow (arrowheads, l). Scale bars are 50 µm (l); 100 µm (k); 500 µm (a–j).