HI-6) Miroslav Pohanka1* Oto Pavlis1 Kamil Kuca1 Jiri Pikula2

Tularemia Progression and it Modulation Including Mortality Remission and Enhancing of Immune System Response Using Asoxime (HI-6) Miroslav Pohanka1* Oto Pavlis1 Kamil Kuca1 Jiri Pikula2

1 Faculty of Military Health Sciences, University of Defence, Trebesska 1575, 50001 Hradec Kralove, Czech Republic 2 Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho 1/3, 612 42 Brno, Czech Republic

KEY WORDS: Francisella tularensis; HI-6; HI-6 in the experiment beginning and then inflammation; oxidative stress; cholinergic the next day. Mice were sacrificed after five anti-inflammatory pathway. days. Plasma, spleen and liver were sam- ABSTRACT pled. In the separate experiment, tularemia caused mortality was assessed with and Objective: without of asoxime application. Francisella tularensis is an intracellular Results and Conclusions: pathogen causing tularemia disease. Immune Regarding to oxidative damage of liver and system action against tularemia is limited spleen, asoxime altered lipid peroxidation due to lipopolysaccharide covering bacterial in liver and significantly reduced oxidative cell. Cholinergic anti-inflammatory pathway damage in spleens. We also proved signifi- is a link between parasympathetic nervous cant increase of plasmatic antibodies level, system and macrophage assisted immunity. decrease of IL6 and steady level of IFNg. Asoxime (also known as HI-6) is a com- Mice treated with asoxime had reduced pound implicated in regulation of acetylcho- mortality when compared to the infected and linesterase as well as acetylcholine recep- untreated ones. The best protective index tors. We hypothesize suitability of asoxime was calculated 2.6 for asoxime doses 2.09 to modulate tularemia progression. and 20.9 mg/kg. Asoxime can be considered Procedure and experiment design: as a compound reducing detrimental impact Laboratory mice BALB/c were infected with of tularemia. Effect of asoxime on choliner- F. tularensis LVS strain and challenged by gic anti-inflammatory pathway and overall application of 209 µg/kg to 209 mg/kg of practical effect is discussed.

76 Vol. 10, No. 1 2012 • Intern J Appl Res Vet Med. Introduction asoxime are suspected to modify functional Francisealla tularensis is a gram negative properties of the nicotinic (nAChR) as bacterium and causative agent of tulare- well as muscarinic (mAChR) acetylcholine mia. The disease manifestation is related to receptors. 17-19 The immune system is as- modes of transmission. The ulceroglandular sociated with parasympathetic regulation by form typically arises after the host is bitten the cholinergic anti-inflammatory pathway by a tick harbouring the agent or in humans (CAP) through a7 nAChR localized on handling infected animals. 1,2 However, macrophages. 20 Pharmacological stimula- tularemia can also elicit oropharyngeal, ocu- tion of CAP is considered to be beneficial loglandular, septic and pneumonic manifes- in the septic shock owing to suppression of tations. 3-5 Several symptoms accompany the pro-inflammatory cytokines overproduction. onset of infection. However, after approxi- 21 Since asoxime is considered a nAChR in- mately three days patients develop unspe- hibitor and its pharmacodynamics allows for cific flu-like symptoms in the first phase.6 only partial penetration through the blood Lymph node enlargement is typical for tula- brain barrier22, maximum effects on blood remia; on the other hand, high temperature receptors can be expected. In this study, en- and fastness of infection progression is quite hancement of macrophages by suppression diverse for individual case reports7. of CAP by asoxime is a hypothesized way of Vaccine strains Moscow, 15 and 155 tularemia progression modulation. derived from F. tularensis holarctica were Material and Methods prepared and tested in the former Soviet Microorganism Union. Live vaccine strain (LVS) was de- Francisella tularensis LVS (ATCC 29684) 8 veloped from strains 15 and 155 in 1950´s. was cryopreserved in liquid nitrogen and At present, there is no licensed vaccine for subcultured before experiments. Bacterium tularemia in the USA or European Union. was cultivated on McLeod agar supple- The efficacy of vaccination with killed F. mented with bovine haemoglobin and Iso tularensis cells is poor since the lipopolysac- VitaleX (Becton-Dickinson, San Jose, CA, charide (LPS) antigen does not trigger an USA) as described previously. 23 Cells were extensive inflammatory response, stimulate harvested after two days of cultivation and cells of the immune system or bind to toll suspended into saline solution and washed 9,10 like receptor - TLR. On the other hand, by centrifugation (2,000×g; 10 minutes). 11 Lavine at al. claimed protective antibody- The cell concentration was estimated by mediated immunity arising after vaccina- calibrated cell density meter (WPA, Cam- tion with heat-killed LVS. Therapy for tularemia is available because streptomycin, Figure 1: HI-6 structure. doxycycline, gentamicin, tetracyclines and quinolones are effective antibiotics used in humans. 12-14 Figure 1 Effects of asoxime on tularemia progression were evaluated in the present study. Asox- ime (HI-6; see figure 1) belongs to potent drugs, the so-called oxime reactivators, that are employed for reactivation of acetylcho- linesterase (AChE) in cases of inhibition by nerve agents 15 and it was recognized to suppress some side effects of drugs as tested for cytostatic agent irinotecan. 16 On the other hand, oxime reactivators including

Intern J Appl Res Vet Med • Vol. 10, No. 1, 2012. 77 bridge, UK) and confirmed by a cultivation saline: 100 mg tissue per one ml of PBS. test two days later. Tissues were mixed 8,000 RPM for 1 minute Animals and blood processing using Ultra-Turrax (Ika Werke, Staufen, A total of 70 female BALB/c mice (BioTest, Germany). The experiment was repeated Konarovice, Czech Republic) were divided with groups 1 - 6 with infection dose 107 into 7 groups of 10 animals. Mice were kept CFU/ml. Mice were observed for pertinent in an air conditioned room (22±2oC) with mortality until symptomatic manifestation of controlled humidity (50±10%) and light (7 tularemia become extinct. a.m to 7 p.m.) within vivarium of the Centre Experiments were permitted and super- of Biological Defence in Techonin (Czech vised by the ethical committee of Centre of Republic). Food and water was supplied Biological Defence (Techonin, Czech Re- ad libitum. In the beginning of experiment, public) and all responsible personnel were mice were eight week old and weighing licensed and skilled for the experiments. 24 g. F. tularensis LVS (LVS hereinafter in Ex vivo assays the text) was adjusted up to 106 CFU/ml. Malondialdehyde (MDA), a marker of oxi- LVS as well as asoxime (Sigma-Aldrich; dative stress, was assayed as thiobarbituric St.Louis, MO, USA) were suspended in acid reactive substance (TBARS) in spleen saline solution prior to the application. The and liver. The measuring protocol was ad- groups were following: opted from the previous experiments. 24 Low 1. 100 µl of LVS; 100 µl saline solu- molecular weight antioxidants (LMWAs) tion were assayed as the ferric reducing anti- 2. 100 µl of LVS; 100 µl asoxime oxidant power (FRAP) as described previ- 50 mg/ml (dose 209 mg/kg of body ously [25, 26]. The level of antibodies was weight) estimated using solid phase extraction (SPE) 3. 100 µl of LVS; 100 µl asoxime 5 using CBindTM L (Sigma-Aldrich) filled mg/ml (20.9 mg/kg) column with subsequent estimation of eluted proteins by the Bradford method according 4. 100 µl of LVS; 100 µl asoxime 500 to previous experiments. 23,27-28 Antioxidant µg/ml (2.09 mg/kg) properties of asoxime were examined by 5. 100 µl of LVS; 100 µl asoxime 50 FRAP method in a way as mentioned above µg/ml (209 µg/kg) and by quenching of 2,2-diphenyl-1-pic- 6. 100 µl of saline solution; 100 µl rylhydrazyl radical (DPPH) using modified asoxime 50 mg/ml (209 mg/kg) experiment according Wozniak et al. 29 7. 2×100 µl of saline solution Asoxime was tested in concentrations 10-6 Saline solution, LVS and asoxime solu- - 10-2 M and compared to standard antioxi- tions were administered subcutaneously dant Trolox (Sigma-Aldrich). Interleukin 6 in the area of the pelvic limb. Asoxime (IL6) and interferon g (IFNg) were assessed was administered one hour after LVS. The using “Murine IL-6 Eli-pair kit” and “Mouse second round of asoxime, respectively saline IFN g Eli-pair kit” (Abcam, Cambridge, solution for groups 1 and 7, application was MA, USA) in compliance with provided in the above-mentioned doses on day two of protocols. experiment. Mice were sacrificed under CO2 Statistical analysis anaesthesia and blood was collected from Software Origin 8 (OriginLab Corporation, cervical vessels on day five of experiment. Northampton, MA, USA) was used for data Fresh blood was let to clot for 30 minutes processing. Significance of differences be- at 4ºC and centrifuged at 500×g for 10 tween groups was estimated using one-way minutes. Freshly prepared serum was used analysis of variance with Scheffe test. The immediately or kept at -80ºC. Spleen and significance was recalculated for two prob- liver were added into phosphate buffered abilities levels P = 0.05 and P = 0.01 for the

78 Vol. 10, No. 1 2012 • Intern J Appl Res Vet Med. Table 1: Assessment of thiobarbituric acid reactive substances (TBARS) and ferric reducing antioxidant power (FRAP) in organs of experimental animals Experimental group 1 2 3 4 5 6 7 Assay Organ

3.37±0.12 2.27±0.27 2.10±0.28 2.41±0.10 2.53±0.20 2.93±0.22 3.21±0.15 (*/-) (*/*) Liver

1.83±0.04 1.48±0.10 1.49±0.14 1.36±0.20 1.48±0.11 1.37±0.14 1.59±0.13 (*) (**/-) (**/-) (**/*) (**/-) (**/*) TBARS (µmol/g) Spleen

4.13±0.11 3.12±0.07 3.32±0.12 3.51±0.10 3.77±0.08 3.09±0.11 3.19±0.16 (*) (*/-) (*/-) (*/-) (*/-) (*) Liver

2.52±0.05 2.64±0.15 2.56±0.06 2.58±0.21 2.68±0.11 2.87±0.10 3.21±0.15 (*) (*/-) FRAP (µmol/g) FRAP Spleen

Error indicates standard error of mean. One and two asterisks represent significance (ANOVA with Scheffe test) at probability level P = 0.05 (*) and P = 0.01 (**) against group 1 (numerator), respectively group 7 (denominator). Asterisks at group 1 indicate significance to control (group 7). group size of eight specimens. values were the second examined parameter. Results We recognized significant (P > 0.05) deple- tion of antioxidants in spleen and increasing In the first round, MDA levels in spleen and in liver in animals infected with tularemia liver as well as spleen and liver antioxidant when compared to the controls. Asoxime potency by FRAP (table 1) were estimated. administration had insignificant impact on Infected mice were compared with the FRAP value in spleen but livers of animals healthy unexposed to asoxime one (group in groups 2 - 4 had decreased FRAP to the 7). MDA in group 1 was found significantly value insignificant to controls. increased in spleen (0.01

0.05) influenced with tularemia had significantly (P ≤ 0.01) only in animals exposed to the two upper increased antibodies levels when compared doses of asoxime. with the control group 7. The antibodies Table 1 ranged from 15.5 ±0.5 (group 1) to 17.2±0.3 mg/ml. Although there was not found a Total level of LMWAs indicated by FRAP significant difference between antibodies

Intern J Appl Res Vet Med • Vol. 10, No. 1, 2012. 79 Table 2: Plasma immunochemical markers

Marker Experimental group 1 2 3 4 5 6 7 Ig (g/l) 15.5±0.4 16.1±0.4 16.3±0.2 16.3±0.3 17.2±0.2 11.8±0.3 12.5±0.3 (*) (-/*) (-/*) (-/*) (*/*) (*/-) IFNg (ng/l) 159±14 144±8 142±9 149±11 143±8 2.83±0.27 2.58±0.78 (**) (-/*) (-/*) (-/*) (-/*) (**/-) IL6 (ng/l) 67.7±2.6 63.9±6.3 56.7±6.4 49.9±3.5 46.7±2.0 2.83±1.1 2.58±2.2 (**) (-/*) (-/*) (-/*) (*/*) (**/-)

Error indicates standard error of mean. One and two asterisks represent significance (ANOVA with Scheffe test) at probability level P = 0.05 (*) and P = 0.01 (**) against group 1 (numerator), respectively group 7 (denominator). Asterisks at group 1 indicate significance to control (group 7). levels in group 1 and groups 2-4, the group asoxime antioxidant properties did not 5 challenged with asoxime 209 µg/kg has proved any antoxidant potency in the significantly (P ≤ 0.01) increased antibodies selected concentration range 10-6 - 10-2 against the group 1. Asoxime administered M. No significant increasing of antioxidant to healthy animals (group 6) had no signifi- properties were found when compared to the cant effect when compared with the control saline solution. Functionality of tests (posi- group 7. Alterations in IFNg and IL6 had tive control) was examined using standard similar tendencies. The both markers were antioxidant Trolox. significantly (0.01

80 Vol. 10, No. 1 2012 • Intern J Appl Res Vet Med. liver enzymes and decreasing LMWAs. 31 In impact, as further discussed bellow, should this experiment, a less virulent LVS strain be expected. was used in this study and blood was col- F. tularensis proliferates within mac- lected from mice infected with a sub-lethal rophages despite the presence of reactive dose. The increased FRAP value in livers forms of oxygen. 37 T lymphocytes are of mice suffering from tularemia can be necessary for enhancing the immune system. attributed to spontaneous protection against Moreover, the lipopolysaccharide (LPS) oxidative stress and decreased FRAP level from F. tularensis does not trigger inflamma- in spleen of tularemia infected mice to the tory response so the innate immunity is of uncovered stress. 32 LMWA can be increased lower efficacy. 9 Due to this fact, stimulation in serum due to hyperuricemia; however, it of the innate immunity could be beneficial is not typical for tularemia in an extensive for the early phase sustained tularemia. IL17 scale. 30 The MDA level appoints at oxida- is necessary for the activation of T lympho- tive stress burden probably caused by pyrop- cytes and in this way it helps macrophages tosis of infected macrophages and conse- to kill phagocyted F. tularensis. 38 Restricted quent release of reactive oxygen species.33,34 growth of F. tularensis in macrophages is It should be concisely mentioned that F. also expected due to IFNγ39. Pharmacologi- tularensis is widely resistant to oxidative cal administration of IFNγ or TNFa can stress within the phagosome by induction of be beneficial for tularemia progress resolv- protective enzymes such as superoxide dis- ing. 40 In a similar way, toll like receptor 3 mutase. 35 Significant reduction of the MDA stimulated with synthetic ligand polyinosine value in spleen is the most interesting find- - polycytosine can protect from tularemia ing and it could be correlated to the reduced pathogenesis as demonstrated on pulmonary mortality of infected mice. However, it is tularemia in BALB/c mice. 41 We found no not clear whether the reduction is caused by significant alteration in IFNγ after asoxime reduced pyroptosis or by another regula- application. IL6 was influenced more exten- tive mechanism. The asoxime impact was sively than IFNγ. IL6 was decreasing in a re- more specific to spleen as the livers were ciprocal proportionality to the asoxime dose. not extensively influenced by asoxime. It is Immunoglobulin levels were influenced in noteworthy to say that MDA level in liver a similar way as IL6: they increased in a nearly two times higher in all tested ani- reciprocal mode to the asoxime dose. Em- mals including control. It refers to fact, that phasising the fact that the markers were as- MDA is produced in body due to oxidative sessed five days post infection, it seems that phosphorylation and other basic physiologi- asoxime has potency to accelerate specific cal pathways. 36 In liver, the FRAP level in immune response to the innate one. Im- infected animals does not clearly correspond provement of mortality confirms benefits of with asoxime administration. The opposite asoxime application into tularemia infected situation was found in spleen. Tularemia in- organisms and practical importance of these fected animals had exhausted LMWAs level findings can be expected. in spleen. Lower doses of asoxime caused Our hypothesis is that there is competi- shift of LMWA that were closer to the value tive protection of a7 nAChR from stimula- in health animals. It is not clear whether tion through acetylcholine which enhances the improved redox balance was caused immunity and elevates the survival probabil- by suppression of pyroptosis or enhancing ity during tularemia. However, it is prob- of antioxidant potency. When considered able that asoxime has ambivalent effects in overall redox situation in liver and spleen, comparison with other compounds such as liver were not exposed to the extensive neostigmine which modulate CAP by inhibi- oxidative stress due to tularemia. FRAP tion of AChE only and cause accumulation method and DPPH tests excluded asoxime of acetylcholine in blood42. Another drug impact as an antioxidant so rather regulative

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