Reduced acetylcholinesterase activity downregulates peripheral and central inflammation during glucocorticoid resistance induced by chronic restraint stress and systemic lipopolysaccharide challenge in male mice

Stress sensitizes the neuroinflammatory response to immunogenic challenge and associated behavioral changes in rodents. GlucocorticoidS (GCs) have been well known for their immunosuppressive and anti-inflammatory properties. However, recent advances have uncovered situations wherein they have opposite effects, especially when activated immune cells show resistance to circulating GCs. Under these circumstances, studying the role of the recently described ‘cholinergic anti-inflammatory pathway’ was of considerable interest. In this study, we investigated the level of serum C-reactive protein (CRP), cytokines and reactive oxygen and nitrogen species and antioxidant enzyme activities in the liver, brain and adrenal gland following LPS administration in stressed mice. Hypothalamic acetyl cholinesterase (AChE) enzyme activity and the expression of heat shock proteins 70 and 90, superoxide dismutase-1 and cycloxygenase-2 proteins in the hypothalamus were estimated by immunobloting. Behavioural changes were observed on an elevated plus maze and in an open field. Our results suggest that there exists a synergistic effect between inflammation and stress only when the stress exposure is acute in nature. Immune activation following chronic stress, downregulated inflammation, in spite of the resistant endocrine response to inflammation, via the newly described cholinergic anti-inflammatory pathway. Thus, it indicates that acute immune activation during chronic stress may be beneficial for the host to maintain homeostasis.

Presence of Toll Like Receptor-2 in spleen, lymph node and thymus of Swiss albino mice and its modulation by Staphylococcus aureus and bacterial lipopolysaccharide.

Toll-like receptors (TLR) are a family of pattern recognition receptors identifying pathogen associated molecular patterns (PAMPs). They play a critical role in the innate immune response during the initial interaction between the infecting microorganism and phagocytic cells. Here, we verified the presence of TLR-2 in spleen, lymph node and thymus of Swiss albino mice and their modulation after infection with Staphylococcus aureus and Lipopolysaccharide (LPS) challenge. It was seen that TLR-2 gene transcribed to its respective mRNA on S. aureus infection, in thymus, spleen and lymph node of mice but their levels and mode of expression varied. When challenged with LPS no prominent changes in the expression of TLR-2 receptor was observed but its expression increased gradually with time in the thymus, spleen and lymph node of S. aureus infected mice. TLR-2 expression was also found enhanced in infected splenic macrophages. By studying the serum cytokine profile the functionality of the receptor was measured. The results indicate the presence of TLR-2 in thymus, spleen and lymph node of Swiss albino strain of mice and that they are modulated by S. aureus.

Ampicillin alone and in combination with riboflavin modulates Staphylococcus aureus infection induced septic arthritis in mice.

Effects of ampicillin (Amp) in combination with riboflavin on septic arthritis in mice infected with Staphylococcus aureus have been reported. Ampicillin was given at 100 mg/kg after 24 h of infection, followed by riboflavin (Ribo) at 20 mg/kg body wt, after 2 h of Amp treatment. Mice were sacrificed at 3, 9, 15 days post infection (dpi). Combined treatment of infected mice with ampicillin and riboflavin eradicated the bacteria from blood, spleen and synovial tissue and showed a significant gross reduction in arthritis, reduced serum levels of TNF- and IFN-. S. aureus infected mice exhibited higher synovial TNF- and IL-6, which was also reduced by ampicillin and riboflavin treatment. S. aureus infected mice showed a disturbed antioxidant status measured in terms of cellular anti-oxidants like reduced glutathione and anti-oxidant enzymes such as superoxide dismutase and catalase and were ameliorated when the animals were co-treated with ampicillin along with riboflavin. Results of the study showed that combined treatment with anti-oxidant and antibiotic may protect from staphylococcal arthritis and may ameliorate oxidative stress caused by S. aureus infection.

Effects of IL-18 and IL-10 pre-treatment on the alteration of endogenous cytokines in liver and spleen of mice with experimental endotoxemia.

Mechanisms of interleukin-18 (IL-18) and interleukin-10 (IL-10) in lipopolysaccharide (LPS) induced endotoxemia are not clear; their protective role is being investigated so that they may effectively modulate the host cytokine levels during endotoxemia. The aim of the study was to evaluate protective effects of IL-18 and IL-10 in experimentally induced endotoxemia in mice correlating the changes in tissue anti-oxidant enzymes and circulating cytokines. Liver injury was determined by estimation of serum glutamate oxalate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT), serum nitric oxide (NOx), hepatic anti-oxidant enzyme and cytokine content in LPS (250 g/kg) induced endotoxemic mice receiving either IL-18 (500 ng/mouse) or IL-10 (600 ng/mouse) treatment. Mice (87% of IL-10 treated and 74% of IL-18 treated) survived when administered prior to LPS challenge. Pre-treatment of mice with either IL-10 or IL-18 followed by LPS, lead to reduction in SGPT and SGOT level, serum NOx, and altered hepatic anti-oxidant enzymes activity and myeloperoxidase activity than the only LPS treated group. Marked reduction in the amounts of LPS-induced hepatic and splenic TNF- content has been observed after IL-10 pre-treatment. Results suggested that attenuating the induction of TNF- and IFN- and subsequent induction of nitric oxide formation in response to LPS may in part account for efficient protection by IL-18 and IL-10 in the reduction of LPS-induced liver injury.

Staphylococcal catalase regulates its virulence and induces arthritis in catalase deficient mice.

To figure out whether in vivo expression of Staphylococcal catalase could correlate with the virulence and pathogenicity of the bacteria in the catalase deficient Swiss albino mice. 3 Amino 1, 2, 4 triazole (ATZ) (2 mg/g body wt) treated catalase deficient mice were infected with virulent S. aureus and bacterial burden, antioxidant enzyme levels were estimated after 3, 5 and 10 days of infection. Arthritic scores and levels of serum uric acid in mice were also determined. ATZ treatment was found to have slowed down the clearance of bacteria from blood and their rapid elimination from spleen. Increased tissue catalase activities in the spleen and liver of ATZ pre-treated mice even after 5 days of infection suggested its bacterial origin. It was further verified by zymographic analysis. Increased swelling of joints was observed after 5 days of infection. Uric acid level was found lesser in ATZ treated mice. ATZ treatment slowed the bacterial passage from blood with a lower tissue anti-oxidant enzymes leading to induction of joint inflammation.

Effects of soluble antigen-induced immune cell activation on steroidogenesis in murine lymphoid organ.

The effect of soluble antigenic (bovine serum albumin, BSA) stimulation to induce steroidogenesis in murine lymphoid organs with concomitant changes in proinflammatory or inflammatory cytokine levels and its implication in the alteration of T-cell response was studied in the mice. Male Swiss albino mice (6-8 weeks old) with average body weight (20 +/- 4 g) were randomly assigned to 3 groups and injected with BSA in presence and absence of Freund's complete or incomplete adjuvant, whereas the control group received only saline. After 3 weeks, animals were sacrificed, and serums as well as lymphoid organs were collected. From the lymphoid tissue homogenate, the activities of steroidogenic enzymes and corticosterone and cytokine levels of the serum were estimated. Steroidogenic enzyme activities in murine lymphoid organs, as well as the pro-inflammatory and inflammatory cytokines levels in serum increased after Freund's complete adjuvant-emulsified BSA administration, as compared to control. The serum corticosterone and serum cytokine profile were also elevated. Results suggested that soluble protein antigen (BSA) administration stimulated steroidogenesis in murine lymphoid tissues and rise in the pro-inflammatory or inflammatory cytokine levels might indicate monocyte recruitment as well as TH1 activation.

Modulation of interleukin-8 receptor expression by lipopolysaccharide (LPS) and phorbol myristate acetate (PMA) in human peripheral monocytes--a preliminary study.

Interleukin-8, a monocyte derived neutrophil chemotactic agent is known to play as a key mediator in the pathogenesis of a large number of neutrophil driven inflammatory diseases. Since the cytokine activates the target cells through a cell surface receptor, study of the regulation of IL-8 receptor expression in monocytes is very important. We found that two very known modulators, lipopolysaccharide (LPS) in presence of homologous serum and Phorbol myristate acetate (PMA) resulted in induction of IL-8 receptor by 100-120% and 75-125% respectively within 1 h in monocytes. Based on the inhibitory effect of cycloheximide, actinomycin-D we may suggest that PMA and LPS could upregulate IL-8 receptor in monocytes through denovo protein synthesis. Prior incubation of polymixin B and anti-CD-14 antibody to the monocytes and subsequent stimulation of the cells with ser.act.LPS resulted in > 90% inhibition of IL-8 binding. Scatchard analysis showed that estimated receptor number in control cell was 7,500 per cell and it increased to 15,500 per cell in ser.act.LPS stimulated cell. The receptor number in PMA stimulated cells was 13,000 per cell. Chemical cross-linking of the IL-8 receptor with 125I labelled IL-8 in the ser.act.LPS and PMA stimulated cells-indicated that the signals at 59 kD were considerably increased with respect to control. A correlation between LPS and ser.act.LPS induced upregulation of IL-8 receptor expression has been shown. The study with bacterial product and co-carcinogenic agent thus provides information about the differential expression of IL-8 receptor for sustained IL-8 mediated biological response.