Effect of one night of sleep loss on changes in tumor necrosis factor alpha (TNF-α) levels in healthy men

Total sleep deprivation in humans is associated with increased daytime sleepiness, decreased performance, elevations in inflammatory cytokines, and hormonal/metabolic disturbances.To assess the effects of 40 h of total sleep deprivation (TSD) under constant and well controlled conditions, on plasma levels of TNF-α and its receptor (TNFR1), interleukin-6 (IL-6), cortisol and C-reactive protein (CRP), sleepiness and performance, 12 healthy men (29 ± 3 years) participated in a 5-days sleep deprivation experiment (two control nights followed by a night of sleep loss and one recovery night). Between 0800 and 2300 (i.e. between 25 and 40 h of sleep deprivation), a serial of blood sampling, multiple sleep latency, subjective levels of sleepiness and reaction time tests were completed before (day 2: D2) and after (day 4: D4) one night of sleep loss. We showed that an acute sleep deprivation (i.e. after 34 and 37 h of sleep deprivation) induced a significant increase in TNF-α (P < 0.01), but there were no significant changes in TNFR1, IL-6, cortisol and CRP. In conclusion, our study in which constant and controlled experimental conditions were realized with healthy subjects and in absence of psychological or physical stressors, an acute total sleep deprivation (from 34 h) was sufficient to induce secretion of pro-inflammatory cytokine such as TNF-α, a marker more described in chronic sleep restriction or deprivation and as mediators of excessive sleepiness in humans in pathological conditions.     

REM sleep deprivation of rats induces acute phase response in liver

REM sleep is essential for maintenance of body physiology and its deprivation is fatal. We observed that the levels of ALT and AST enzymes and pro-inflammatory cytokines like IL-1β, IL-6 and IL-12 circulating in the blood of REM sleep deprived rats increased in proportion to the extent of sleep loss. But in contrast the levels of IFN-γ and a ∼200 kDa protein, identified by N-terminal sequencing to be alpha-1-inhibitor-3(A1I3), decreased significantly. Quantitative PCR analysis confirmed that REM sleep deprivation down regulates AII3 gene and up regulates IL1 β, IL6 and their respective receptors gene expression in the liver initiating its inflammation.     

The Pro-inflammatory Role of TGFβ1: A Paradox?

TGFβ1 was initially identified as a potent chemotactic cytokine to initiate inflammation, but the autoimmune phenotype seen in TGFβ1 knockout mice reversed the dogma of TGFβ1 being a pro-inflammatory cytokine to predominantly an immune suppressor. The discovery of the role of TGFβ1 in Th17 cell activation once again revealed the pro-inflammatory effect of TGFβ1. We developed K5.TGFβ1 mice with latent human TGFβ1 overexpression targeted to epidermal keratinocytes by keratin 5. These transgenic mice developed significant skin inflammation. Further studies revealed that inflammation severity correlated with switching TGFβ1 transgene expression on and off, and genome wide expression profiling revealed striking similarities between K5.TGFβ1 skin and human psoriasis, a Th1/Th17-associated inflammatory skin disease. Our recent study reveals that treatments alleviating inflammatory skin phenotypes in this mouse model reduced Th17 cells, and antibodies against IL-17 also lessen the inflammatory phenotype. Examination of inflammatory cytokines/chemokines affected by TGFβ1 revealed predominantly Th1-, Th17-related cytokines in K5.TGFβ1 skin. However, the finding that K5.TGFβ1 mice also express Th2-associated inflammatory cytokines under certain pathological conditions raises the possibility that deregulated TGFβ signaling is involved in more than one inflammatory disease. Furthermore, activation of both Th1/Th17 cells and regulatory T cells (Tregs) by TGFβ1 reversely regulated by IL-6 highlights the dual role of TGFβ1 in regulating inflammation, a dynamic, context and organ specific process. This review focuses on the role of TGFβ1 in inflammatory skin diseases.     

Lack of phospholipase C-delta1 induces skin inflammation

Phospholipase C (PLC) is a key enzyme in phosphoinositide signaling. We previously generated PLC-delta1 knockout (KO) mice and found that these mice showed remarkable hair loss caused by abnormalities in hair follicle structures. Here we show that the skin of PLC-delta1 KO mice displays typical inflammatory phenotypes, including increased dermal cellularity, leukocyte infiltration, and expression of pro-inflammatory cytokines. In addition, exogenously expressed PLC-delta1 attenuates lipopolysaccharide-induced expression of IL-1beta, a pro-inflammatory cytokine, in an enzymatic activity-dependent manner. Furthermore, suppression of skin inflammation by anti-inflammatory reagents cured the epidermal hyperplasia in PLC-delta1 KO mice. Taken together, these results indicate that lack of PLC-delta1 induces skin inflammation and that the epidermal hyperplasia in PLC-delta1 KO mice is caused by skin inflammation. Our results also suggest that PLC-delta1 regulates homeostasis of the immune system in skin.     

Differential inflammatory activation of IL-6 (-/-) astrocytes

IL-6 is a major immunomodulatory cytokine with neuroprotective activity. The absence of interleukin-6 (IL-6) results in increased vulnerability of dopaminergic neurons to the neurotoxicant, MPTP, and a compromised reactive microgliosis. To determine how astrogliosis may contribute to nigrostriatal degeneration in IL-6 (-/-) mice, the inflammatory profiles of astrocytes of IL-6 genotype were compared. Fourteen cytokines and four chemokines were simultaneously assayed in the supernatants of LPS-stimulated primary astrocyte cultures. In a time course of 6, 18 and 48 h and LPS stimulations of 0, 0.1, 1, 10 and 100 ng/ml, IL-6 (-/-) astrocytes secreted significantly greater amounts of the pro-inflammatory cytokines IL-1alpha, IL-1beta and TNFalpha than did IL-6 (+/+) cells. Elevated levels of IL-10 and IL-12p40 were only detected at 48 h post-stimulation with greater IL-10 in IL-6 (-/-) supernatants and greater IL-12p40 in IL-6 (+/+) supernatants. IL-6 (+/+) astrocytes produced more G-CSF and GM-CSF when compared with IL-6 (-/-) astrocytes. Chemokine levels were greater in supernatants of IL-6 (+/+) astrocytes than IL-6 (-/-) cells prior to 48 h post-stimulation. At that time, higher levels of MIP-1alpha were maintained in IL-6 (+/+) supernatant, while similar levels of MCP-1 in supernatants of both IL-6 (+/+) and IL-6 (-/-) cells were measured. Additionally, LPS (100 ng/ml) resulted in greater levels of KC and Rantes in IL-6 (-/-) astrocyte supernatants compared with IL-6 (+/+) supernatants at that time. These results suggest that the autocrine modulatory activities of IL-6 affect multiple cytokine secretory pathways, which could participate in neurodegenerative processes.     

Interleukin-12 but not interleukin-18 is required for immunity to Trypanosoma cruzi in mice

Protective immunity to the parasite Trypanosoma cruzi in mice depends on a pro-inflammatory T cell response involving the production of interferon-gamma (IFN-gamma). In conjunction with interleukin-12 (IL-12), IL-18 promotes the synthesis of IFN-gamma and a T helper type 1 immune response. We investigated the requirements of IL-12 and IL-18 in murine T. cruzi infection by use of C57BL/6 mice genetically deficient in either cytokine. IL-12p40(-/-) mice succumbed to infection at doses of 100 parasites, whereas IL-18(-/-) and wild-type mice resisted infectious doses up to 1000 parasites to the same extent. Levels of parasitemia were comparable between the latter groups, as were tissue parasite burdens according to quantitative real-time PCR. In contrast, IL-12p40(-/-) mice displayed vastly increased levels of parasites both in blood and in tissue. IFN-gamma concentrations in the serum of infected mice and in supernatants of splenocytes stimulated in vitro were decreased in IL-18(-/-) mice, whereas in IL-12p40(-/-) mice, IFN-gamma was undetectable in the serum and drastically reduced in cell supernatants. Levels of IL-12 production were generally comparable between wild-type and IL-18(-/-) mice, as were levels of IL-4, IL-2 and nitric oxide. Thus, the requirement for endogenous pro-inflammatory cytokines for a protective murine immune response against T. cruzi is satisfied by the expression of IL-12, while IL-18 is dispensable.     

Clinical, pathological and immunological features of psoriatic-like lesions affecting keratin 14-vascular endothelial growth factor transgenic mice

Up-regulation of vascular endothelial growth factor (VEGF) plays a primary role in the pathogenesis of psoriasis. Transgenic mice over-expressing VEGF under the Keratin 14 (K14) promoter develop an inflammatory skin condition with many of the pathobiological features of human psoriasis. In this work, the development of spontaneous psoriatic-like dermatitis in K14-VEGF transgenic mice was monitored from week 6 to week 44 and skin lesions were characterized clinically (application of a clinical score system comparable to the human Psoriasis Area and Severity Index), microscopically (histopathology, leukocyte subset and neoangiogensis) and immunologically (evaluation of local and systemic cytokine/chemokine profiles). Based on PASI score system, three progressive clinical phases were identified: mild acute (8-14 weeks of age), moderate subacute (15-21 weeks of age) and severe chronic-active (22-44 weeks of age) dermatitis. Microscopically, skin lesions consisted of progressive proliferative psoriatic-like dermatitis dominated by dermo-epidermal infiltrates of CD3-positive lymphocytes, an increased number of mast cells and neoangiogenesis. Both local and systemic up-regulation of pro-inflammatory (IL-12, TNF-alpha, IL-6, MCP-1 and IL-8) and regulatory (IL-10) cytokines/chemokines was observed, mainly during the later stages of disease development. The results obtained in this study further confirm the central role of VEGF over-expression in the development of psoriatic-like dermatitis. Similarly to what is reported for human psoriasis, both the local and systemic immunologic profiles observed in K14-VEGF transgenic mice suggest that a combined Th1 and Th17 response may be implicated in lesion development. The identification of three progressive stages of disease, each with peculiar clinicopathological features, renders the K14-VEGF transgenic mouse a valuable model to study novel immunotherapies for psoriasis.     

Lipopolysaccharide-induced liver apoptosis is increased in interleukin-10 knockout mice

Although IL-10 down-regulates pro-inflammatory cytokine secretion by hepatic Kupffer cells, the mechanisms underlying its hepatoprotective effects are not fully clear. This study tested the hypothesis that IL-10 protects the liver against pro-inflammatory cytokines by counteracting their pro-apoptotic effects. Wild type and IL-10 knockout mice were treated with bacterial lipopolysaccharide and sacrificed 1, 4, 8, and 12 h later. Plasma ALT activity was measured as a marker of liver injury. Liver pathology and TUNEL response were assessed by histology. Plasma levels and whole liver mRNA levels were measured for TNF-alpha, IL-1 beta, TGF-beta1, IL-10, and their respective receptors. Hepatic mRNA levels were measured for several pro-apoptotic adaptors/regulators, including FasL, Fas receptor, FADD, TRADD, Bad, Bak, Bax, and Bcl-X(S), and anti-apoptotic regulators, including Bcl-w, Bcl-X(L), Bcl-2, and Bfl-1. Caspase-3 activity in the liver was determined as well as immunohistochemistry for IL-1RII, TGF-betaRII and Fas receptor. At all time points the livers from IL-10 knockout mice displayed a significantly increased number of apoptotic nuclei compared to wild type mice. Changes in plasma cytokine levels and their liver mRNA levels were consistent with suppression by IL-10 of pro-inflammatory cytokine secretion. In addition, pro-inflammatory cytokine receptor mRNA levels (TNF-alpha, TGF-beta, and IL-1 beta) were markedly up-regulated by LPS at all time points in IL-10 knockout mice as compared to wild type mice. Expression of the pro-inflammatory cytokine receptor IL-1RII was similarly increased as shown by immunostaining. The mRNA levels of a typical pro-apoptotic cytokine, TRAIL, were increased and LPS also up-regulated the mRNA expression of other apoptotic factors to a larger extent in IL-10 knockout mice than in their wild type counterparts, suggestive of an IL-10 anti-apoptotic effect. In the livers of knockout mice, markedly increased caspase-3 activity was already evident at the 1-h time point following LPS administration, while in the wild type animals this increase was delayed. Immunostaining also indicated that LPS increased hepatic expression of the pro-apoptotic receptors Fas and TGF-betaRII in IL-10 knockout mice. The data presented in this study show that: (i) IL-10 modulates not only the secretion of pro-inflammatory cytokines, but also the receptors of these cytokines, and ii) IL-10 protects the liver against LPS-induced injury at least in part by counteracting pro-inflammatory cytokine-induced liver apoptosis.     

Cytokines as potential therapeutic targets for inflammatory skin diseases

A network of pro-inflammatory cytokines is a central feature in the pathophysiology of cutaneous inflammatory diseases. Thus, the delineation of precise roles for particular cytokines and the development of cytokine-directed therapeutics have become areas of intense investigation. While anti-TNF therapeutics have proven to be effective for the treatment of psoriasis, clinical investigations have now begun with other cytokine-directed therapies, such as those targeting IFN-g, IL-12p40, and IL-18. In addition to therapeutics that target cytokines directly, strategies that target cytokine signaling pathways are in development too. In this short review, we summarize key findings from a recent workshop on cytokines as potential therapeutic targets for inflammatory skin diseases.     

Effects of the Linear Fragments of Beta-(1→3)-Glucans on Cytokine Production <Emphasis Type="Italic">in vitro</Emphasis>

Beta-glucans, homopolysaccharides composed of 3,6-branching β-(1→3)-D-glucan chains, attract great interest as inducers of cytokine synthesis. In this work, we studied the ability of linear fragments of beta-glucan chains to activate cytokine synthesis. Synthetic nona-β-(1→3)-D-glucoside (SO) representing a linear fragment of beta-glucan chain, endotoxin (ED), and natural β-(1→3)-D-glucan (GL) were tested for their role as inducers of cytokines in whole peripheral blood cultures collected from 17 individuals. The concentrations of IL-12p70, IFN-γ, IL-2, IL-10, IL-8, IL-6, IL-4, IL-5, IL-1β, TNF-α, and TNF-β were measured in the supernatants after 2, 24, and 48 h of cell culturing. SO, ED, and GL stim- ulated production of pro-inflammatory IFN-γ, IL-1β, IL-2, IL-6, IL-8, TNF-α and anti-inflammatory IL-10. The high- est levels of biosynthesis after stimulation with SO were registered for IL-6, IL-8, and TNF-α. SO stimulated production of all cytokines (except IFN-γ) to a lesser extent than ED and GL. The IFN-γ/IL-10 (Th1/Th2) ratios after 24 and 48 h of culturing were 3.1 and 7.5 for SO; 0.03 and 0.1 for GL; and 0.06 and 0.2 for ED, respectively. The results indicate that lin- ear fragments of beta-glucans cause a more pronounced shift of immune response towards the pro-inflammatory (Th1) type than beta-glucan itself.     

Sex-related effects of sleep deprivation on depressive- and anxiety-like behaviors in mice

Anxiety and depressive symptoms are generated after paradoxical sleep deprivation (PSD). However, it is not clear whether PSD produces differential effects between females and males. The aim of this study was to assess the effect of PSD on anxiety- and depressive-like behaviors between sexes. Male and female BALB/c mice were divided in three groups: the control group, the 48-h PSD group and the 96-h PSD group. Immediately after PSD protocols, the forced swimming and open field test were applied. Sucrose consumption test was used to evaluate the middle-term effect of PSD. We found that corticosterone serum levels showed significant differences in the 96-h PSD females as compared to 96-h PSD males. In the open-field test, the 48-h and 96-h PSD females spent more time at the periphery of the field, and showed high locomotion as compared to males. In the elevated plus maze, the 48-h PSD females spent more time in closed arms than males, which is compatible with anxiety-like behavior. The forced swim test indicated that the 96-h PSD males spent more time swimming as compared to the 96-h PSD females. Remarkably, the 96-h PSD males had lower sucrose intake than the 96-h PSD females, which suggest that male mice have proclivity to develop a persistent depressive-like behavior late after PSD. In conclusion, male mice showed a significant trend to depressive-like behaviors late after sleep deprivation. Conversely, female have a strong tendency to display anxiety- and depressive-like behaviors immediately after sleep deprivation.     

Intravenous infection of virulent shigellae causes fulminant hepatitis in mice

Shigella spp. are pathogenic bacteria responsible for bacillary dysentery in humans. The major lesions in colonic mucosa are intense inflammation with apoptosis of macrophages and release of pro-inflammatory cytokines. The study of shigellosis is hindered by the natural resistance of rodents to oral infection with Shigella. Therefore, animal models exploit other routes of infection. Here, we describe a novel murine model in which animals receive shigellae via the caudal vein. Mice infected with 5 x 10(6) (LD(50)) virulent shigellae died at 48 h post infection, whereas animals receiving non-invasive mutants survived. The liver is the main target of infection, where shigellae induce microgranuloma formation. In mice infected with invasive bacteria, high frequency of apoptotic cells is observed within hepatic microgranulomas along with significant levels of mRNA for pro-inflammatory cytokines such as IL-1beta, IL-18, IL-12 and IFN-gamma. Moreover, in the blood of these animals high levels of IL-6 and transaminases are detected. Our results demonstrate the intravenous model is suitable for pathogenicity studies and useful to explore the immune response after Shigella infection.     

Correlation of IL-12, IL-22, and IL-23 in patients with psoriasis and metabolic syndrome. Preliminary report

BackgroundPsoriasis is an autoimmune skin disease characterised by proliferation of keratinocytes, primarily due to cytokines Th1 and Th17. This profile is involved in pathogenesis of metabolic syndrome, a frequently found comorbidity in patients with psoriasis.ObjectiveIn this study we determine the correlation of levels of pro-inflammatory cytokines TNF-α, IL-23, IL-12, and IL-22 in patients with psoriasis with and without metabolic syndrome and clinically healthy controls.MethodsWe included 55 patients with plaque psoriasis: 30 with metabolic syndrome (PPMS), 25 without metabolic syndrome (PP), 15 healthy subjects (HS) and 15 with metabolic syndrome (MS). Quantification of serum levels of IL-12, TNF-α, IL-22, and IL-23 was done by ELISA.ResultsWe observed that serum levels of IL-12 were more elevated in PP group, while the lowest levels of TNF-α were seen in HS group. IL-22 was found to be higher in PP than in PPMS (p < 0.05). PP patients with PASI scores rating as severe showed higher levels of IL-12. TNF-α level analysis showed significant differences in HS group compared with the others; levels of this cytokine were lower in patients with PP and moderate PASI scores than in MS group (p < 0.05). We found no correlation between cytokine levels and psoriasis or between cytokines and PASI scores. In PP group, a positive correlation was observed between IL-23 and fasting glucose (r = 0.432, p < 0.05), as well as a negative correlation between IL-23, IL-22, and IL-12 versus waist circumference (r = −0.504, r = −0.556 and r = −0.511, respectively; p < 0.05).ConclusionsPsoriasis is not just a skin disorder, but rather a condition with systemic implications, with intervention of pro-inflammatory cytokines that contribute to metabolic syndrome and other comorbidities, which in turn increases the risk of developing cardiovascular disease.     

A comparative study of the immune modulating properties of antifibrinolytics in cardiac surgery

PurposeAntifibrinolytics, used in cardiac surgery to abate postoperative blood loss, share anti-inflammatory properties by suppression of pro-inflammatory D-dimer and plasmin levels. Additional drug specific immune modulating qualities are often mentioned in the discussion on which antifibrinolytic can best be used. To determine the extent and relevance of these effects, we investigated cytokine and growth factor plasma levels in cardiac surgery patients randomized to receive either tranexamic acid, aprotinin, or placebo. Corticosteroid-treated patients served to put the effects in perspective.MethodsUsing a biochip immunoassay, plasma of 36 cardiac surgery patients was quantified for 12 cytokines and growth factors, assessed preoperatively and 6, 12, 24, and 48 h after the start of cardiopulmonary bypass. Eight patients were treated with tranexamic acid, nine with aprotinin, and nine received placebo. Ten placebo-treated patients received corticosteroids.ResultsIL-1ß, IL-6, IL-8, IL-10, IFN-γ, TNF-α, VEGF, MCP-1, and EGF plasma concentrations significantly changed over time across all patients. Aprotinin-treated patients showed decreased pro-inflammatory TNF-α and peak MCP-1 plasma levels when compared with placebo. However, corticosteroids attenuated the inflammatory response to a much larger extent, lowering postoperative IL-6, IL-10, IFN-γ, and VEGF concentrations also.ConclusionsAprotinin attenuates postoperative pro-inflammatory levels TNF-α and MCP-1 whereas tranexamic acid does not. The majority of plasma proteins studied, however, were not affected by the use of antifibrinolytics when compared with placebo. A clinically relevant common anti-inflammatory effect through inhibition of fibrinolysis seems therefore unlikely.     

Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17

Interleukin (IL)-17 is a pro-inflammatory cytokine that is produced by activated T cells. Despite increasing evidence that high levels of IL-17 are associated with several chronic inflammatory diseases including rheumatoid arthritis, psoriasis, and multiple sclerosis, the regulation of its expression is not well characterized. We observe that IL-17 production is increased in response to the recently described cytokine IL-23. We present evidence that murine IL-23, which is produced by activated dendritic cells, acts on memory T cells, resulting in elevated IL-17 secretion. IL-23 also induced expression of the related cytokine IL-17F. IL-23 is a heterodimeric cytokine and shares a subunit, p40, with IL-12. In contrast to IL-23, IL-12 had only marginal effects on IL-17 production. These data suggest that during a secondary immune response, IL-23 can promote an activation state with features distinct from the well characterized Th1 and Th2 profiles.     

Impact of sex on hyperalgesia induced by sleep loss

This study evaluated the impact of sex on the short term consequences of different periods of sleep deprivation and the effect of the respective sleep recovery periods on nociceptive responses. Male and female C57BL/6J mice were assigned to the following groups: paradoxical sleep deprived (PSD) for 72 h, sleep restricted (SR) for 15 days, exposed to respective recovery periods for 24 h, or untreated home-cage controls (CTRL). Mice were submitted to a noxious thermal stimulus to evaluate their nociceptive response after PSD, SR, or recovery periods. Blood was collected for hormonal analysis. The nociceptive response was significantly lower in PSD and SR mice compared to CTRL animals, regardless of the sex. However, SR females had a lower paw withdrawal threshold than males. Sleep recovery was able to restore normal nociceptive sensitivity after PSD in both sexes. The hyperalgesia induced by SR was not reversed by sleep rebound. In females, low concentrations of estradiol were found after SR, and these concentrations continued to decrease after 24 hours of sleep recovery. The PSD male mice exhibited higher concentrations of corticosterone than the CTRL and SR male mice. Corticosterone levels were not affected by SR. Our study revealed that PSD and SR induce hyperalgesia in mice. The SR groups showed marked changes in the nociceptive response, and the females were more sensitive to these alterations. This finding indicates that, although different periods of sleep deprivation change the nociceptive sensitivity in male and female mice, sex could influence hyperalgesia induced by chronic sleep loss.     

Pretreatment with granulocyte-colony stimulating factor decreases lipopolysaccharide-induced interferon-gamma production in mice in association with the production of interleukin-18

We studied the effects of pretreatment with granulocyte-colony stimulating factor (G-CSF) on the production of pro- and anti-inflammatory cytokines induced by lipopolysaccharide (LPS). Mice received G-CSF or control saline once a day for 7 days or once at 1 h before the injection of LPS. Cytokines were measured by enzyme-linked immunosorbent assay or antibody-based electrochemiluminescence assay and cytokine mRNA was measured by RNAse protection assay. Mice pretreated with G-CSF for 7 days before LPS had lower serum levels of LPS-induced interferon-gamma (IFN-gamma) and higher levels of interleukin (IL)-6 and IL-10 than controls. G-CSF-pretreated mice also had lower mRNA levels of IFN-gamma and higher mRNA levels of IL-6 and IL-10 in the spleen and/or liver than controls. G-CSF-pretreated mice had serum levels of tumor necrosis factor, IL-12 p70 and IL-12 p40 similar to controls. G-CSF-pretreated mice had lower levels of spleen IL-18 than controls-serum IL-18 being undetectable in mice after LPS-and lower levels of IL-18 mRNA in the spleen. Mice pretreated with G-CSF 1 h before LPS had lower levels of serum IFN-gamma and spleen IL-18 than controls. G-CSF pretreatment alters the expression of LPS-induced cytokines with a decrease in pro-inflammatory IFN-gamma and an increase in anti-inflammatory IL-6 and IL-10. G-CSF decrease of IL-18 production may be a major mechanism explaining the effects of G-CSF on the production of IFN-gamma.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> flagellin stimulates pro-inflammatory immune response

Flagellin, a principal component of bacterial flagella, is a virulence factor that is recognized by the innate immune system. Recognition of flagellin by innate immune receptors stimulates the production of cytokines necessary for the development of effective immunity. Here, we demonstrated that the intranasal (i.n.) instillation of different amount of Escherichia coli K-12 flagellin preparation (0.5, 1, 2, 4 μg) in BALB/c mice induced pro-inflammatory immune response. Instillation i.n. of 1 μg of flagellin induced the maximum expression of interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) mRNA and production of pro-inflammatory cytokines (IL-1β, TNF-α and IL-6) in mice lungs. The same dose of flagellin induced neutrophil polymorphonuclear cells infiltration in peribronchial and perivascular regions. High number of neutrophil in bronchoalveolar lavage fluid was found at 24 h after i.n. instillation of flagellin (1 μg). These findings were concomitant with the maximum production of myeloperoxidase and nitric oxide in mice lungs. Present study showed that the maximum pro-inflammatory mediator levels were found when mice instilled i.n. with 1 μg E. coli flagellin. The amount of flagellin of E. coli K-12 that achieve the maximum stimulation of mucosal pro-inflammatory immune response in mice lungs was explored in this study.