Acetylcholine and an acetylcholinesterase inhibitor neostigmine can aggravate tularemia progress in BALB/c mice

The present experiment was aimed at assessing the application of neostigmine, an acetylcholinesterase (AChE) pseudo-irreversible inhibitor with poor penetration through the hematoencephalitic barrier, and the neurotransmitter acetylcholine (ACh). The experiment was done to evaluate their ability to modulate an infectious disease: tularemia. Mice infected with Franciselle tularensis and exposed to either ACh or neostigmine had a higher mortality and spleen bacterial burden when compared to infected mice exposed to saline solution only. The activated cholinergic anti-inflammatory pathway suppressed pathways necessary for tularemia resolution. Administration of AChE inhibitors to the individuals suffering from tularemia is contra-indicatory. Drugs based on AChE inhibition should be restricted when tularemia or disease with a similar pathogenesis is suspected.


Introduction
The immunomodulatory role of acetylcholine (ACh) has been considered for a long time (Kaufer et al., 2003). The connection of the nervus vagus to the cholinergic antiinflammatory pathway (CAP) as a specific regulation of the innate immune system by the parasympaticus was first recognized by Tracey and co-workers (Tracey, 2002). CAP was first referd to as reflex-like anti-inflammatory pathway, then the term CAP was introduced. The pathway is based on α7 nicotine acetylcholine receptor (nAChR) on the surface of macrophages and ACh is released into the blood system from the nervus vagus termination (Tracey, 2009). In the macrophages, the activated CAP inhibits the production of tumor necrosis factor alpha (TNF-α) and other pro-inflammatory intermediates (Wang et al., 2003). Miroslav Pohanka, Oto Pavlis, Hana Svobodova, Jiri Pikula

Bacterium
Francisella tularensis LVS (ATCC 29684) was cultivated on McLeod agar supplemented with bovine hemoglobin and Iso VitaleX (Becton-Dickinson, San Jose, CA, USA) and processed as described previously (Pohanka et al., 2007). Cells were harvested after two days of cultivation and were washed by saline solution using centrifugation 2,000×g for 10 minutes. Concentration of fresh cells was estimated using a cell density meter (WPA, Cambridge, UK) and confirmed by a cultivation test.

Animals and tissues processing
Fiftysix three-month old female BALB/c mice (BioTest, Konarovice, Czech Republic) were divided into 7 groups. Each group contsisted of 8 animals. The mice were kept in an air conditioned room with steady temperature (22±2°C) and humidity (50±10%). The artificial light was adjusted at periods from 7 a.m to 7 p.m. The experiment was carried out within the vivarium of the Centre of Biological Defence in Techonin (Czech Republic) and was approved by the central ethical committee (Ministry of Defense, Czech Republic). Food and water was supplied ad libitum during the whole experiment. In the beginning of the experiment, the mice were eight weeks old and weighed on average 20 g. F. tularensis LVS was suspended in saline solution and adjusted to 10 5 colony forming units (CFU)/ml. Ft LVS as well as neostigmine and ACh (Sigma-Aldrich; St.Louis, MO, USA) were suspended in saline solution prior to application. The groups were as follows: • 100 μl of Ft suspension; 100 μl of saline solution • 100 μl of Ft suspension; 100 μl of neostigmine 40 mg/l, dose 0.2 mg/kg body weight • 100 μl of Ft suspension; 100 μl of acetylcholine 60 mg/l, dose 0.3 mg/kg body weight • 100 μl saline solution; 100 μl of neostigmine 40 mg/l, dose 0.2 mg/kg body weight • 100 μl of saline solution; 100 μl of acetylcholine 60 mg/l, dose 0.3 mg/kg body weight • 100 μl of saline solution for two administrations. The solutions were administered subcutaneously in the area of the pelvic limb. Neostigmine, ACh and saline solution were applied one hour after Ft suspension. Tularemia was confirmed in all infected animals by the appearance of typical disease signs.
After five days, mice were sacrificed under CO 2 anesthesia. Spleens were collected and homogenized for cultivation in order to estimate the bacterial burden. The mortality experiment was carried out in the same way as given above. The dose of Ft was 100 μl 10 8 CFU/ml. All other parameters were unchanged.

Statistical analysis
Origin 8 (OriginLab Corporation, Northampton, MA, USA) was used for data processing throughout the experiments and performed for descriptive as well as inferential statistics. Significance of differences between the groups tested was estimated using one-way analysis of variance with Tukey´s test. The significance was recalculated for two probability levels p=0.05 as well as p=0.01 for the group size n=8.

Results
The bacterial burden of the spleen was assayed immediately after the animals had been sacrificed. In compliance with expectations, the animals that were not infected with tularemia had no positive cultivation proof. The animals infected with tularemia had on average content 5.50×10 4 Ft CFU per spleen. Animals infected and simultaneously exposed to ACh or neostigmine had significantly (p≤0.01) increased Ft levels. The spleens from animals exposed to neostigmine had on average of 1.38×10 5 CFU. The highest Ft content was found after ACh administration: 3.95×10 5 CFU. The data are shown in Table 1.
The mortality test compared the impact of the compounds tested on survival of tularemia infected animals ( Figure 1) No mortality was observed in mice treated only with saline solution (control), ACh, or neostigmine. Infection with Ft caused 40% mortality. Co-application of ACh resulted in 50% mortality (comparison to Ft group: Table 1. Bacterial burden in spleen of mice exposed to tularemia (Ft), tularemia with neostigmine (Ft + neo), and tularemia with acetylcholine (Ft + ACh).

Ft
Ft + neo Ft + ACh Chi Square 0.833; df=1; p=0.361) and of neostigmine in 60% mortality (Chi Square 3.33; df=1; p=0.067). The observed mortality occurred from the third to fourth day post infection. No mortality occurred before and after that time.

Discussion
The multiple factors investigated during the experiments pointed to some interesting processes accompanying tularemia and/or CAP stimulation. The pyroptosis cell-death bears signs of necrosis and pro-inflammatory reaction (Bergbaken & Cookson, 2009;Kepp et al., 2010). The stress markers arising during tularemia were also described in previous experiments (Pohanka et al., 2009;Bandouchova et al., 2009a). Generally, activation of CAP is expected to ameliorate inflammation induced pathogeneses as the direct impact on tissues can be diminished.
On the other hand, activation of CAP can reduce the ability of the immune system to fight the pathogen. Both ACh and neostigmine were administered in safe doses. The dose of ACh corrresponded to the mammalian physiological level (Fujii et al., 1997). The neostigmine dose was correlated to the common medium amount per kilogram used in human medicine (Kopman and Eikermann, 2009). The most significant impact of neostigmine as well as of ACh was recognized in the bacterial burden and tularemia induced mortality. The bacterial burden was significantly increased in the spleen both after neostigmine and ACh administration. The bacterial burden corresponds to the organism's sensitivity to tularemia (Bandouchova et al., 2009b) and an elevated bacterial burden should deductively correspond to higher mortality. In full agreement with the hypothesis, increased mortality was found after ACh as well as neostigmine administration.
CAP is linked to suppression of inflammation. When it is stimulated, the inflammation signaled by C-reactive protein and TNF-α is decreased (Jae et al., 2009). The hypothesis set in the experiment was based on the question whether administration of compounds directly stimulating nAChR, Ach in the presaent study, or compounds inhibiting AChE, represented by neostigmine, and thus elevating endogenous blood ACh level, could modulate tularemia progression. Neostigmine was chosen on purpose for the fact that it does not cross the blood-brain barrier (Yamamoto et al., 1995). The effect was not clearly comprehensible as parasympathetic rather than sympathetic response should be expected due to acetylcholine-evoked relaxation (Sotnikova et al., 2009). The drug for Alzheimer´s disease treatment, Donepazil, has the same effect as it reduces stress and increases the ACh level even during exposition to bacterial liopolysaccharide (Tyagi et al., 2010). On the other hand, the recognized oxidative stress after ACh administration could be triggered by another mechanism, not involved in ACh regulative function.
Stimulation of CAP and suppression of related cytokines production is well expected (Rosas-Ballina et al., 2009). The primary question was to assess whether tularemia can be influenced by stimulation of CAP. The response was positive. Mortality and bacterial burden confirmed that stimulation of CAP in the first stage of tularemia is a negative process as the activated CAP does not allow simple activation of macrophages. The explanation is in full compliance with experiments of other authors. IL-1β deficient mice have a lower probability to survive tularemia (Fernandes-Alnemri et al., 2010). Cytokines such as IFN-γ and TNF-α as well as T H1lymphocytes are necessary to resolve the disease (Elkins et al., 2009;Lin et al., 2009). On the other hand, Ft needs to survive in macrophages and proliferate there in the phagosome (Bell et al., 2010). Thus activated CAP could suppress pathways necessary for tularemia resolution. Administration of AChE inhibitors to individuals suffering from tularemia is contraindicatory. Drugs based on AChE should be restricted when tularemia is suspected. On the contraty, inhibition of CAP can be hypothesized as an appropriate approach since a positive effect can be expected.

Conclusions
CAP seems to be a relevant regulatory tool influencing the immune system. Unsubstantial stimulation of CAP can deteriorate proliferation of the intracellular pathogen F. tularensis. Drugs based on AChE inhibition or nAChR stimulation are not applicable to individuals suspected to suffer from tularemia. Their impact is significantly contraindicatory. On the other hand, blockade of the nAChR is hypothesized as a suitable tool to be assessed in further experiments regarding tularemia treatment.