Leptospira interrogans induces uterine inflammatory responses and abnormal expression of extracellular matrix proteins in dogs

Highlights

• Leptospira interrogans caused abortion in dogs.

• ECM is related to reproduction.

• L. interrogans-infected dogs showed abnormal level of ECM.

Abstract

Leptospira interrogans (L. interrogans), a worldwide zoonosis, infect humans and animals. In dogs, four syndromes caused by leptospirosis have been identified: icteric, hemorrhagic, uremic (Stuttgart disease) and reproductive (abortion and premature or weak pups), and also it caused inflammation. Extracellular matrix (ECM) is a complex mixture of matrix molecules that is crucial to the reproduction. Both inflammatory response and ECM are closed relative to reproductive. The aim of this study was to clarify how L. interrogans affected the uterus of dogs, by focusing on the inflammatory responses, and ECM expression in dogs uterine tissue infected by L. interrogans. In the present study, 27 dogs were divided into 3 groups, intrauterine infusion with L. interrogans, to make uterine infection, sterile EMJH, and normal saline as a control, respectively. The uteruses were removed by surgical operation in 10, 20, and 30 days, respectively. The methods of histopathological analysis, ELISA, Western blot and qPCR were used. The results showed that L. interrogans induced significantly inflammatory responses, which were characterized by inflammatory cellular infiltration and high expression levels of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in uterine tissue of these dogs. Furthermore, L. interrogans strongly down-regulated the expression of ECM (collagens (CL) IV, fibronectins (FN) and laminins (LN)) in mRNA and protein levels. These data indicated that strongly inflammatory responses, and abnormal regulation of ECM might contribute to the proliferation of dogs infected by L. interrogans.

Introduction

Leptospirosis, a worldwide reemerging zoonosis, caused by Leptospira interrogans (L. interrogans) which is a Gram-negative bacteria has emerged as a public problem throughout the world, especially in tropical areas [1], [2]. As a typical reservoir animal, rodents carry L. interrogans in their kidneys and pass it out with the urine, contaminating the environment [3]. Humans and animals, including dogs, cattle and swine, become infected by leptospires in contaminated water, soil, urine or other tissues, they enter the host via skin abrasions or mucous membranes, disseminate rapidly via the blood, and invade the host tissues and organs [4]. Leptospirosis main clinical manifestations are fever, jaundice, renal and hepatic insufficiency, pulmonary hemorrhage and reproductive failure. Since dogs as very popular pets for human being are also susceptible to leptospires [5], the infection by leptospires could be a great threat to human health. Four syndromes caused by leptospirosis in dogs have been identified: icteric, hemorrhagic, uremic (Stuttgart disease) and reproductive (abortion and premature or weak pups) [6]. And reproductive seems like a more severe problem, studies showed that bacterial uterine infection is a common disease that develops in 25% of all intact female dogs and characterized by mainly Gram-negative infection in combination with severe local and systemic inflammation [7], [8]. Moreover, the reproductive may cause other health problem, such as the dangers of infertility. Currently, there exists are limited studies related to the reproduction of dogs by leptospirosis [9], and the mechanism of reproduction is unknown.

Previous research indicated that ECM in uterus and reproduction had very intimate relationships [10]. ECM is primarily to mediate cell proliferation, movement, differentiation, embryo implantation and pregnancy, which consists of a complex mixture of matrix, including the fibronectins (FN), Type I and IV collagens (CL I and IV), laminins (LN) and proteoglycans that assemble into fibrils or other complex macromolecular arrays [11]. These components of ECM are organized as macromolecules containing subunits and different regions in order to interact with each other also with extracellular domains of cell surface receptors [12]. Studies confirmed that CL IV, FN, and LN have a close relationship with pregnancy [13]. CL IV is the major protein component of the basal membrane and is also one of the principal structures of the uterine ECM at the fetal–maternal interface [14], [15]. FN and LN have much similarity on biological characters, and they have some distinct function on cell behavior. While FN can modulate cell adhesion and migration during embryogenesis, LN strongly promotes cell growth, morphogenesis and differentiation, alters the cell function, and plays a key role in the early stages of the epithelial histogenesis [16]. It is clearly that the regulation disorder of ECM could lead to fail reproduction. The study of our lab showed that Mycoplasma bovis was triggered endometrial inflammatory response and abnormal expression of ECM in uterus of cows [17]. In addition, the major surface protein LipL32, LigA, and LigB of L. interrogans has been reported to bind to host ECM [6], [18], but it is not clear the role of L. interrogans in the regulation of ECM, especially the ECM in uterus. Moreover, L. interrogans could cause inflammation in canine [19] and the inflammatory response is correlation with uterine disease, such as endometritis and abortion [20].

The objective of the present study was to clarify how L. interrogans affect the uterus, by focusing on the inflammatory responses, and ECM expression in dogs uterine tissue infected by L. interrogans, and to provide a reliable evidence for the potential mechanism of reproductive (abortion and premature or weak pups), clinical diagnosis, and treatment in dogs.