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Acute Brucella Melitensis M16 Infection Model in Mice Treated with Tumor Necrosis Factor-Alpha Inhibitors

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Acute Brucella Melitensis M16 Infection Model in Mice Treated with Tumor Necrosis Factor-Alpha Inhibitors Original Article Acute Brucella melitensis M16 infection model in mice treated with tumor necrosis factor-alpha inhibitors Murat Kutlu1, Çağrı Ergin2, Nilay Şen-Türk3, Selda Sayin-Kutlu1, Orçun Zorbozan2, Şerife Akalın1, Barboros Şahin4, Veli Çobankara5, Neşe Demirkan3 1 Department of Infectious Diseases and Clinical Microbiology, Pamukkale University, Faculty of Medicine, Denizli, Turkey 2 Department of Medical Microbiology, Pamukkale University, Faculty of Medicine, Denizli, Turkey 3 Department of Pathology, Pamukkale University, Faculty of Medicine, Denizli, Turkey 4 Animal Research Laboratory, Pamukkale University, Denizli, Turkey 5 Department of Internal Medicine, Section of Rheumatology, Pamukkale University, Faculty of Medicine, Denizli, Turkey Abstract Introduction: There is limited data in the literature about brucellosis related to an intracellular pathogen and anti-tumor necrosis factor alpha (anti-TNFα) medication. The aim of this study was to evaluate acute Brucella infections in mice receiving anti-TNFα drug treatment. Methodology: Anti-TNFα drugs were injected in mice on the first and fifth days of the study, after which the mice were infected with B. melitensis M16 strain. Mice were sacrificed on the fourteenth day after infection. Bacterial loads in the liver and spleen were defined, and histopathological changes were evaluated. Results: Neither the liver nor the spleen showed an increased bacterial load in all anti-TNFα drug groups when compared to a non-treated, infected group. The most significant histopathological findings were neutrophil infiltrations in the red pulp of the spleen and apoptotic cells with hepatocellular pleomorphism in the liver. There was no significant difference among the groups in terms of previously reported histopathological findings, such as extramedullary hematopoiesis and granuloma formation. Conclusions: There were no differences in hepatic and splenic bacterial load and granuloma formation, which indicate worsening of the acute Brucella infection in mice; in other words, anti-TNFα treatment did not exacerbate the acute Brucella spp. infection in mice. Key words: experimental brucellosis; anti-TNF-alpha drug; etanercept; infliximab. J Infect Dev Ctries 2015; 9(2):141-148. doi:10.3855/jidc.5155 (Received 21 April 2014 – Accepted 09 Spetember 2014) Copyright © 2015 Kutlu et al. This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction Brucella infection [4-6]. TNFα has a critical role in Anti-tumor necrosis factor alpha (TNFα) drugs are macrophage activation, apoptosis, proinflammatory widely used for rheumatoid arthritis, ankylosing cytokine release, and granuloma formation. It is spondylitis, inflammatory bowel disease, psoriasis, required for bactericidal effects and clearance of psoriatic arthritis, and juvenile chronic arthritis. Brucella infection [7-9]. Although it was reported that However, anti-TNFα drug therapy is associated with TNFα was not secreted by mouse macrophages increased risk of infection, including non-typhoidal infected with B. melitensis [10], TNFα gene salmonellosis, listeriosis, pneumocystosis, and other expression and induction was demonstrated in in vitro fungal or viral infections, but Mycobacterium and in vivo studies conducted with mice macrophage, tuberculosis infection incidence is especially increased splenocyte, and dendritic cells [11-13]. Additionally, in patients who are treated with these drugs [1,2]. the increased TNFα serum level was shown with B. Brucellosis is still endemic in some parts of the abortus infection, but not with B. melitensis [14]. world, and approximately 500,000 new cases are Although brucellosis is an endemic disease and there reported annually [3]. Brucella spp. are intracellular is a widespread use of anti-TNFα drugs for different bacteria that release cytokines such as IFN-gamma, indications, reactivation of brucellosis can be expected IL-2, and TNFα. Activated macrophages and dendritic more frequently. However, there are only three case cells are required for the immune response against reports about brucellosis reactivation due to anti-TNFα Kutlu et al. – Brucella infection in mice treated with anti-TNFα J Infect Dev Ctries 2015; 9(2):141-148. drug therapy [15-17]. In this study, we aimed to placed in sterile Petri dishes with 10 mL of saline. The investigate the responses to acute Brucella infection in organs were homogenized for quantitative bacterial mice receiving adalimumab (ADA), etanercept (ETN), load by mincing them with two no. 15 scalpels and and infliximab (IFX) by evaluating the splenic and homogenizing by pushing them through the mesh with hepatic bacterial loads as well as histopathological a sterile syringe plunger. Aliquots of 0.1 mL spleen findings. and liver homogenate samples were diluted tenfold serially in PBS. Next, 100 µL homogenates of each Methodology dilution were cultured on HBA plates at 37°C with 5% Bacteria CO2 for five days. The quantitative bacterial load of This study used a standard smooth strain of B. each sample was determined by log conversion. melitensis provided by the Pendik Veterinary Control and Research Institute in Istanbul, Turkey. Histopathology Lyophilized bacteria were suspended in phosphate Tissue samples of the spleen and liver taken from buffered saline (PBS) and cultured on horse blood agar each mouse were fixed in 10% buffered formalin (HBA) plates for three days at 37°C with 5% CO2. placed in paraffin blocks, sectioned at 5 µm, and stained with haematoxylin and eosin for histological Animals, infections, and anti-TNFα drugs evaluation. Two pathologists performed a blinded The study was carried out according to the ethical evaluation of histopathological changes in the spleen guidelines (no. 2011.035) of the Medical Ethics and liver, using the following histopathological Committee of Pamukkale University, Denizli, Turkey. evaluation criteria: A total of 56 BALB/c mice between eight and ten weeks of age, weighing between 30 and 40 grams, Spleen were included in the study. During the experiment Granuloma – 0: absent, 1: micro granuloma, 2: period, six mice were excluded from the analysis due macro granuloma; extramedullary hematopoiesis – 0: to preterm death. The control groups (CG) (non- absent, 1: mild, 2: moderate, 3: severe; lymphoid infected-CG, n = 6; infected-CG, n = 7) and following tissue depletion – 0: absent, 1: mild, 2: moderate, 3: test groups were examined: ADA (adalimumab, n = severe; neutrophil infiltration in the red pulp – 0: 6); ADA + B. melitensis (adalimumab with acute B. absent, 1: present. melitensis infection, n = 6); ETN (etanercept, n = 6); ETN + B.melitensis (etanercept with acute B. Liver melitensis infection, n = 7); IFX (infliximab, n = 6); Granuloma – 0: absent, 1: micro granuloma, 2: and IFX + B. melitensis (infliximab with acute B. macro granuloma; extramedullary hematopoiesis – 0: melitensis infection, n = 6). absent, 1: mild, 2: moderate, 3: severe; apoptotic cells On days 1 and 5 of the experiment period, drugs – 0: absent/minimal, 1: present; portal inflammation – were administered subcutaneously to the ADA (2 0: absent, 1: mild, 2: moderate, 3: severe; perivascular mg/kg), ETN (0.4 mg/kg), and IFX (10 mg/kg) groups, mononuclear infiltration – 0: absent, 1: present; while 100 µL PBS injections were given to the control Kuppfer cell hyperplasia – 0: absent, 1: mild, 2: group. The aim of this study was to evaluate acute moderate, 3: severe; and hepatocellular pleomorphism Brucella infection in mice receiving anti-TNFα drug – 0: absent, 1: present. treatment, so a pre-infection regime was preferred, and the treatment was repeatedly performed as in previous Statistical analysis studies [18-20]. Test groups were intraperitoneally The statistical analysis was carried out using SPSS inoculated with 4x104 colony-forming units (cfu) of B. software version 17. The bacterial load in the spleen melitensis M16 strain on day 5. Optimal doses were and liver were analyzed using the Mann-Whitney U described by previous studies for virulent Brucella, test (two group comparison) and the Kruskal-Wallis and in this study, the infective dose was administered test (multi group comparison). The histopathological as in some other studies on a B. melitensis model findings of the spleen and liver were analyzed by the [11,13,21]. Chi-square and Kruskal-Wallis tests. Statistical significance was set at p < 0.05. Measurement of bacterial load in liver and spleen On day 19 (the 14th day of infection), the spleen and liver were aseptically removed, weighed, and 142 Kutlu et al. – Brucella infection in mice treated with anti-TNFα J Infect Dev Ctries 2015; 9(2):141-148. Results in the red pulp was more prominent in the groups that Quantitative spleen bacterial load were administered anti-TNFα, and there was a Bacterial growth was observed in spleen tissue statistically significant difference between the ADA + cultures of all infected mice. The lowest bacteria B. melitensis and infected-CG groups (p = 0.026) number was in the ETN + B.melitensis group, while (Figure 3). the highest was in the IFX + B. melitensis group (Figure 1). There was no difference in quantitative Liver histopathology splenic bacterial load between the groups receiving Although granuloma was determined in all infected-CG and anti-TNFα. The IFX + B. melitensis samples, extramedullary hematopoiesis was detected group had
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