GPR109A controls neutrophil extracellular traps formation and improve early sepsis by regulating ROS/PAD4/Cit-H3 signal axis.

BACKGROUND: Neutrophil extracellular traps (NETs) is the key means for neutrophils to resist bacterial invasion. Sepsis is a systemic inflammatory response syndrome caused by infection. METHODS: In our study, qRT-PCR was used to detect the gene expression in neutrophils, Western blot was used to detect the protein expression in mouse tissues and neutrophils, flow cytometry was used to detect the purity of neutrophils in the whole blood and immunofluorescence was used to detect the NETs formation. RESULTS: In this study, we analyzed the NETs formation in the blood of patients with sepsis. The results showed that a large number of NETs appeared. And the expression of GPR109A in neutrophils of patients with sepsis was significantly up regulated. Then we collected neutrophils from WT mice and GPR109A-/- mice and found that GPR109A knockout could significantly inhibit the early NETs formation of neutrophils. The results also showed that knockout of GPR109A or inhibition of the NETs formation could increase the inflammatory response of liver, spleen, lung and kidney in mice, thus affecting the disease process of sepsis. Then we observed the death of mice in 16 days. The results showed that inhibiting the NETs formation could significantly affect the early mortality of mice, while knocking out GPR109A could directly affect the mortality of the whole period. CONCLUSIONS: This study confirmed the regulatory effect of GPR109A on early NETs formation for the first time, and provided a new target for the treatment of sepsis.

A lethal model of Leptospira infection in hamster nasal mucosa.

Leptospirosis is a fatal zoonosis caused by contact between skin or a mucosal surface and contaminated soil or water. Hamsters were infected by intraperitoneal injection fto establish experimental leptospirosis, which is not a natural route of infection. There are no reports of nasal mucosal infection in hamsters. In this study, infection of the nasal mucosa was performed to establish a model of natural infection. Both methods of infection can cause lethal models with similar symptoms in the later stages of infection, such as weight loss, blood concentration, increased neutrophils (GRAN), and decreased lymphocytes (LYM) in the blood, severe organ damage and liver function obstruction. The burden of Leptospira in the organs and blood was lower in the mucosal inoculation groups at 1 day after infection. However, mucosal infection induced a higher Leptospira burden in urine than intraperitoneal infection in the late stages of infection. After nasal mucosal infection, antibody levels were higher and lasted longer. These results indicated that the route of nasal mucosal infection is a good choice for studying leptospirosis in hamsters.

IL-10 Deficiency Protects Hamsters from Leptospira Infection.

Leptospirosis is a global zoonotic disease with outcomes ranging from subclinical infection to fatal Weil's syndrome. In addition to antibiotics, some immune activators have shown protective effects against leptospirosis. However, the unclear relationship between Leptospira and cytokines has limited the development of antileptospiral immunomodulators. In this study, the particular role of interleukin-10 (IL-10) in leptospirosis was explored by using IL-10-defective (IL-10-/-) hamsters. After Leptospira infection, an improved survival rate, reduced leptospiral burden, and alleviation of organ lesions were found in IL-10-/- hamsters compared with wild-type (WT) hamsters. In addition, the levels of expression of the IL-1ß, IL-6, and tumor necrosis factor alpha (TNF-α) genes and the level of nitric oxide (NO) were higher in IL-10-/- hamsters than in WT hamsters. Our results indicate that IL-10 deficiency protects hamsters from Leptospira infection.

Norfloxacin suppresses Leptospira-induced inflammation through inhibiting p65 and ERK phosphorylation and NLRP3 inflammasome activation.

Leptospirosis is a worldwide re-emerging zoonosis caused by pathogenic Leptospira. Inflammatory storms induced by Leptospira are the reason to induce immunoparalysis and organ failures. Antibiotics are still the current mainstream treatment for leptospirosis. In addition to their antibacterial action, the immunomodulatory function of antibiotics has been paid more and more attention. In this study, the role of norfloxacin on Leptospira-induced inflammation was investigated. Treatment with norfloxacin down-regulated Leptospira-induced IL-1ß and TNF-α both in vivo and vitro models. Further study showed that norfloxacin inhibited Leptospira-induced phosphorylation of p65 and ERK. Norfloxacin also inhibited the Leptospira-induced NLRP3 inflammasome activation with the increased level of Na/K-ATPase Pump ß1 subunit and decreased level of Kcnk6. These results indicated that norfloxacin suppressed Leptospira-induced inflammation through inhibiting p65 and ERK phosphorylation and NLRP3 inflammasome activation. Norfloxacin may be a potential candidate for suppressing inflammatory storms caused by Leptospira.

Emergency vaccine immunization protects hamsters against acute leptospirosis.

Leptospirosis, caused by pathogenic Leptospira, is a global critical zoonotic disease in terms of mortality and morbidity. Vaccines are often used to prevent leptospirosis. However, few studies have reported the therapeutic effect of a vaccine against Leptospira infection. This study demonstrates the efficacy of the emergency vaccine immunization against acute leptospirosis in hamsters. Treatment with a whole-cell vaccine (Leptospira interrogans serovar Lai) at 24 h post-infection improved the survival rate of hamsters with lower leptospiral burden and minor pathological damage to organs. The vaccine also protected against multiple Leptospira serotypes acute infection. However, the protective effect of the vaccines was lost when beginning treatment at 36 h or 48 h post-infection. These results indicated that vaccines could treat acute leptospirosis in hamsters, but only if immunization is within 24 h after infection.

Dipotassium glycyrrhizinate relieves leptospira-induced nephritis in vitro and in vivo.

Chronic leptospirosis usually occurs during sublethal doses infection of susceptible animal and reservoir host, which typical symptom is interstitial nephritis, and leptospira urine, contaminating the environment and threatening other susceptible animals and humans. Dipotassium glycyrrhizinate (DG) is a replacement for glycyrrhizic acid, which exhibits anti-inflammation, immunomodulation effects. This study is to investigate whether DG relieves leptospira-induced nephritis. In vitro, DG inhibited the leptospira-induced transcription levels of IL-1ß, IL-6, TNF-α, RANTES, MCP-1 and iNOS, and protein levels of IL-1ß and TNF-α, and downregulated NF-κB and MAPK pathway in TCMK-1 cells. In vivo, DG attenuated the kidney histopathological change and downregulated the expression of IL-1ß and TNF-α, as well as reduced kidney leptospiral burden. In summary, DG alleviated leptospira-induced inflammation through inhibitory NF-κB and MAPK pathway, and DG decreased the renal colonization of leptospires in mice.

The pre-activated immune response induced by LPS protects host from leptospirosis.

Leptospirosis is an important global zoonosis caused by pathogenic Leptospira. It is estimated that more than 1 million people are infected by Leptospira each year, and the death toll is about 60,000. Some studies showed that delayed immune response was associated with severe leptospirosis, and TLR4 was very important in the control of leptospirosis. In this study, we aimed to explore the effect of the classical activator (LPS) of TLR4 on leptospirosis in susceptible and resistant hosts. The results showed that LPS pretreatment increased the survival rate of hamsters to 80%. And LPS pre-treatment also significantly reduced the leptospiral load and alleviated the pathological injury in organs of hamsters and mice. The result detected by ELISA in mice showed that the levels of TNF-α and IL-1ß were increased in the LPS-treated group compared to the control group before infection. However, two days after infection, the level of cytokines in LPS group was down-regulated compared with that in control group. In addition, in vitro results showed that LPS pre-treatment enhanced the phagocytosis and bactericidal ability of macrophages on Leptospira. Collectively, our results indicated that the pre-activated immune response induced by LPS enhanced the ability of host against leptospirosis.

Increased inflammation with crude E. coli LPS protects against acute leptospirosis in hamsters.

Leptospirosis is a worldwide zoonotic disease that causes acute kidney injury, liver disease, bleeding disorders, and even death. Treatment of the disease is largely dependent on the use of antibiotics, but recent studies on pathogenesis of leptospirosis have shown that immunomodulation may also be an effective treatment for this disease. Since the delay in inflammation correlates with higher pathogenicity of leptospira, we studied the effect of inducing inflammation on leptospirosis by using TLR4 activator LPS. In accordance with our hypothesis, treatment with LPS protected against leptospirosis by enhancing the inflammatory response in hamsters. The gene expression levels of TLR2, TLR4, NLRP3 and inflammatory factors were higher in LPS-treated group during leptospira infection in hamsters. Although the levels of NO and iNOS were higher in LPS-treated group than in Leptospira-infected group, the protective effect induced by LPS is iNOS-independent. Treatment with LPS induced higher anti-leptospira IgG level than infection with leptospira alone. Then, expressions of costimulatory molecules and maturation markers were analysed. The data showed that treatment with LPS enhanced the expression of CD40, CD80 and CD86. Our results indicate that increased inflammation induced by LPS derived from Escherichia coli (E. coli) protects against leptospirosis in hamsters.

Preliminary Characterization of Dog Derived Pathogenic Strains of Leptospira interrogans Serovar Australis in Nanchang of Jiangxi Province, China.

Leptospirosis is a global zoonotic disease caused by pathogenic Leptospira, and those infected animals will show a variety of clinical symptoms and even death. The discovery of endemic strains is crucial to produce effective vaccines. In this study, we report that a strain of Leptospira, isolated from a dog, is pathogenic. Using MLST analysis, the serovar of isolated Leptospira was identified and found it belongs to Leptospira interrogation Serovar Australis. Then, the virulence of this strain was researched by using hamsters. After infection, all the hamsters died within 4-5 days. Typical pathological changes were found in the liver, kidney, and lung of hamsters. These results all indicated that the isolated Leptospira was pathogenic. Thus, this study facilitates to identifying local Leptospira strains and develop a more targeted canine Leptospira vaccine.

The preventable efficacy of ß-glucan against leptospirosis.

Leptospirosis, caused by pathogenic Leptospira species, has emerged as an important neglected zoonotic disease. Few studies have reported the preventable effects of immunoregulators, except for antibiotics, against leptospirosis. Generally, immunostimulatory agents are considered effective for enhancing innate immune responses. Many studies have found that beta-glucan (ß-glucan) could be a potent and valuable immunostimulant for improving immune responses and controlling diseases. In this study, we investigated the preventable role of ß-glucan against Leptospira infection in hamsters. First, ß-glucan was administered 24 h prior to, during and after infection. The results showed that ß-glucan increased the survival rate to 100%, alleviated tissue injury, and decreased leptospire loads in target organs. Additionally, we found using quantitative real-time PCR that application of ß-glucan significantly enhanced the expression of Toll-like receptor (TLR) 2, interleukin (IL)-1ß and iNOS at 2 dpi (days post infection) and reduced the increase of TLR2, IL-1ß and iNOS induced by Leptospira at 5 dpi. Furthermore, to induce memory immunity, ß-glucan was administered 5 days prior to infection. ß-Glucan also significantly increased the survival rates and ameliorated pathological damage to organs. Moreover, we demonstrated that ß-glucan-trained macrophages exhibited elevated expression of proinflammatory cytokines (IL-1ß and IL-6) in vitro, indicating that ß-glucan induces an enhanced inflammatory response against Leptospira infection. These results indicate that administration of ß-glucan and other immunostimulants could be potential valuable options for the control of Leptospira infection.

Low-dose Norfloxacin-treated leptospires induce less IL-1ß release in J774A.1 cells following discrepant leptospiral gene expression.

Currently, accumulating evidence is challenging subtherapeutic therapy. Low-dose Norfloxacin (Nor) has been reported to suppress the immune response and worsen leptospirosis. In this study, we investigated the influence of low-dose Nor (0.03 µg/ml, 0.06 µg/ml, 0.125 µg/ml) on leptospiral gene expression and analyzed the immunomodulatory effects of low-dose Nor-treated leptospires in J774A.1 cells. To study the expression profiles of low-dose Nor-treated leptospires, we chose LipL71/LipL21 as reference genes determined by the geNorm applet in this experiment. The results showed that low-dose Nor up-regulated the expression of FlaB and inhibited the expression of 16S rRNA, LipL32, LipL41, Loa22, KdpA, and KdpB compared with the untreated leptospires. These results indicated that low-dose Nor could regulate leptospiral gene expression. Using RT-PCR, the gene expression of IL-1ß and TNF-α in J774A.1 cells was detected. Nor-treated leptospires induced higher expression levels of both IL-1ß and TNF-α. However, when analyzed by ELISA, the release of mature IL-1ß was reduced compared with that observed in cells induced with no Nor-treated leptospires, although the TNF-α protein level showed no significant change. Our study indicated that the gene expression of leptospires could be modulated by low-dose Nor, which induced less IL-1ß release in J774A.1 cells.

Doxycycline Attenuates Leptospira-Induced IL-1ß by Suppressing NLRP3 Inflammasome Priming.

Doxycycline (Dox), a semisynthetic antibiotic, has been reported to exert multiple immunomodulatory effects. Treatment with Dox has a satisfactory curative effect against leptospirosis. In addition to its antibacterial action, we supposed that Dox also modulated immune response in controlling leptospira infection. Using J774A.1 mouse macrophages, the effects of Dox on protein and mRNA levels of IL-1ß and TNF-α were investigated after infection with live or sonicated Leptospira interrogans serovar Lai strain Lai (56601). Specifically, the level of IL-1ß but not TNF-α was sharply decreased when treated with Dox in leptospira-infected macrophages. Western blot analysis showed that Dox suppressed the activation of leptospira-induced MAPK and NF-κB signaling pathways. Using NLRP3-deficient and NLRC4-deficient mice, the data showed that the expression of leptospira-induced IL-1ß was mainly dependent on the presence of NLRP3 inflammasome in macrophages. Meanwhile, Dox suppressed leptospira-induced NLRP3 inflammasome priming with the upregulation of the Na/K-ATPase Pump ß1 subunit. The inhibition effect of Dox on IL-1ß was also conspicuous in cells with lipopolysaccharide and ATP stimulation. These results were confirmed in vivo, as peritoneal fluids of mice and organs of hamsters expressed less IL-1ß after treatment of leptospiral infection with Dox. Our results indicated that Dox also modulated immune response to attenuate leptospira-induced IL-1ß by suppressing p38, JNK, p65, and NLRP3 inflammasome priming.

Low-dose norfloxacin and ciprofloxacin therapy worsen leptospirosis in hamster.

Antibiotics play an important role in the treatment of leptospirosis. Many antibiotics at appropriate concentrations improved the survival rate and alleviated tissue injury, while, when dosing strategies fall below subtherapeutic levels, worse therapeutic effects are seen. In the present study, we investigated the efficacy of low-dose norfloxacin (10, 20 and 30 mg/kg) and ciprofloxacin (1, 2 and 5 mg/kg) against leptospirosis in a hamster model using Leptospira interrogans serovar Icterohaemorrhagiae. The histopathology and bacterial loads of target organs (liver, kidney and lung) were also studied by treatment with norfloxacin at the dose of 10 mg/kg in this model. Using RT-PCR, the expression of inflammatory factor IL-1ß and TNF-α was analyzed by comparing the norfloxacin and untreated group. All untreated animals, serving as a negative control, displayed 50% survival rate, while hamsters treated with norfloxacin at the dose of 10 and 20 mg/kg and ciprofloxacin at the dose of 1 and 2 mg/kg showed a lower survival rate than the untreated group. Furthermore, norfloxacin at the dose of 10 mg/kg increased bacterial loads and aggravated tissue injury of target organs. The delayed induction of IL-1ß and TNF-α was found in tissues of norfloxacin group. Our study indicates an increased risk associated with low-dose norfloxacin and ciprofloxacin in leptospirosis.

Efficacy of the Rabbit Polyclonal Anti-leptospira Antibody against Homotype or Heterotype Leptospira Infection in Hamster.

Leptospirosis, caused by Leptospira, is one of the most important of neglected emerging zoonotic diseases that has important impacts on public health worldwide. Polyclonal antibody (pcAb) therapy is a potential method to process a series of pathogens for which there are limited determination of treatment, such as leptospirosis. First, we evaluated the efficacy of pcAb, derived from the sera of rabbits inoculated with Leptospira, against homotype (Leptospira interrogans serovar Lai) or heterotype (Leptospira interrogans serovar Autumnalis) Leptospira infection in a lethal hamster model. The pcAb treatment improved survival compared to the controls. The histopathology's of the infected kidney, liver and lung were also examined by hematoxylin and eosin staining. Using real-time quantitative PCR, we determined that most of the leptospires in the primary organs were almost completely removed by pcAb. In the second experiment, treatments, including antibiotic, pcAb, and combination, were started immediately after occurrence of the first serious sickness mouse in any group. No significant difference in survival rate between pcAb group and antibiotic group was found, but the combination therapy group significantly improved survival rate compared to the others (P<0.05). We conclude that the rabbit pcAb treatment may cure both the homotype and the heterotype lethal Leptospira infections in hamster, and combination therapy improved survival compared to antibiotic group in the late treatment of homotype leptospirosis.

Bovine lactotroph cultures for the study of prolactin synthesis functions.

The aim of this study was to establish a bovine anterior pituitary-derived lactotroph (BAPDL) line that expresses prolactin (PRL) in vitro to study the mechanisms of bovine PRL synthesis and secretion. Immunohistochemistry assay of PRL in the newborn calves' anterior pituitary glands showed that most lactotrophs were located within the superior border of the lateral wings of the anterior pituitary. Tissues of the superior border of the lateral wings of the anterior pituitary were dispersed and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The limiting dilution method was used to establish BAPDL from single cell clone. BAPDL cells constantly expressed mRNAs for PRL and pituitary-specific transcription factor 1 (Pit-1) gene and grew steadily and rapidly in the DMEM supplemented with 10% FBS. PRL immunoreactivity was present in BAPDL at passage 20. The concentration of bovine PRL in BAPDL at passage 20 culture supernatant was decreased to below 35% compared with that in BAPDL at passage 1. The effects of human epidermal growth factor (hEGF) and dopamine (DA) on the expression and secretion of PRL in BAPDL at passage 4 were also investigated. The results are consistent with those of previous studies. Thus, it can be used successfully for studying the mechanisms of stimuli regulating PRL synthesis and release.

Effects of insulin-like growth factor-1 on the assembly and secretion of very low-density lipoproteins in cow hepatocytes in vitro.

Fatty liver is a major metabolic disorder of dairy cows. One important reason is that hepatic very low-density lipoproteins (VLDL) assembly was significant decreased in dairy cows with fatty liver. In addition, the impairment of insulin-like growth factor (IGF)-1 synthesis was involved in the development of fatty liver. Therefore, the objective of this study was to investigate the effects of IGF-1 on the VLDL assembly in cow hepatocytes. In this study, cow hepatocytes were cultured and then transfected with Ad-GFP-IGF-1 (inhibited the IGF-1 expression) and Ad-GFP (negative control), and treated with different concentrations of IGF-1, respectively. The results showed that IGF-1 increased the mRNA abundance of apolipoprotein B100 (ApoB100), apolipoprotein E (ApoE), microsomal triglyceride transfer protein (MTTP), and low-density lipoprotein receptor (LDLR) and then increased the VLDL assembly in cow hepatocytes. Nevertheless, impairment of IGF-1 expression by Ad-GFP-IGF-1 could inhibit above genes expression and VLDL assembly in hepatocytes. Taken together, these results indicate that IGF-1 increases the VLDL assembly and impairment of IGF-1 expression decreases the VLDL assembly in cow hepatocytes.