Mucosal delivery of anti-inflammatory IL-1Ra by sporulating recombinant bacteria
© Porzio et al; licensee BioMed Central Ltd. 2004
Received: 06 July 2004
Accepted: 30 October 2004
Published: 30 October 2004
Mucosal delivery of therapeutic protein drugs or vaccines is actively investigated, in order to improve bioavailability and avoid side effects associated with systemic administration. Orally administered bacteria, engineered to produce anti-inflammatory cytokines (IL-10, IL-1Ra), have shown localised ameliorating effects in inflammatory gastro-intestinal conditions. However, the possible systemic effects of mucosally delivered recombinant bacteria have not been investigated.
B. subtilis was engineered to produce the mature human IL-1 receptor antagonist (IL-1Ra). When recombinant B. subtilis was instilled in the distal colon of rats or rabbits, human IL-1Ra was found both in the intestinal lavage and in the serum of treated animals. The IL-1Ra protein in serum was intact and biologically active. IL-1-induced fever, neutrophilia, hypoglycemia and hypoferremia were inhibited in a dose-dependent fashion by intra-colon administration of IL-1Ra-producing B. subtilis. In the mouse, intra-peritoneal treatment with recombinant B. subtilis could inhibit endotoxin-induced shock and death. Instillation in the rabbit colon of another recombinant B. subtilis strain, which releases bioactive human recombinant IL-1β upon autolysis, could induce fever and eventually death, similarly to parenteral administration of high doses of IL-1β.
A novel system of controlled release of pharmacologically active proteins is described, which exploits bacterial autolysis in a non-permissive environment. Mucosal administration of recombinant B. subtilis causes the release of cytoplasmic recombinant proteins, which can then be found in serum and exert their biological activity in vivo systemically.
The use of recombinant proteins as drugs has deeply modified the therapeutic approach to many severe diseases. However, a variety of practical problems limits the use of biotechnological protein drugs. Stability of the active proteins, need for parenteral administration, and high costs of the final purified materials are among the most significant drawbacks. A way of circumventing these issues is represented by the direct administration of recombinant bacteria, acting simultaneously as cell factory and delivery system for pharmacologically active proteins. This approach has been already extensively experimented for the mucosal delivery of vaccine antigens [1, 2]. In recent years, the local delivery of therapeutic antibodies [3, 4], adjuvant cytokines [5, 6], and anti-inflammatory cytokines [7–9] has been successfully attempted with food-grade bacteria (e.g., Lactococcus lactis, Streptococcus gordonii), although limited to the therapy of localised pathologies (e.g., inflammatory bowel diseases, IBD, in the gastro-intestinal tract).
Among anti-inflammatory strategies, both at systemic and local level, the use of the IL-1 receptor antagonist (IL-1Ra) has received vast attention. IL-1 is a family of cytokines highly active in the modulation of immune amplification and inflammation. The IL-1 family includes two agonist proteins, IL-1α and IL-1β, and one antagonist protein, IL-1Ra. IL-1β is a very potent immunostimulatory and inflammatory cytokine, responsible for initiating and amplifying the host response to invasion. If not properly controlled, IL-1 can cause fever, acute inflammation, tissue destruction, organ failure, and eventually shock and death (reviewed in ). IL-1Ra inhibits IL-1 by acting as a competitive receptor antagonist with no detectable agonist activity, thus representing a natural powerful mechanism to control IL-1-dependent responses and avoid pathological derangement (reviewed in [11, 12]). In experimental animal models, IL-1Ra has demonstrated excellent therapeutic effects against acute and chronic inflammatory pathologies, being also effective at high doses in prolonging survival in endotoxic shock [11–17]. In human trials, IL-1Ra has been administered to patients with septic shock, rheumatoid arthritis, graft-versus-host disease, and multiple sclerosis (reviewed in [11, 12, 16]). While only a modest benefit was achieved in patients with septic shock [11, 12, 16, 18], IL-1Ra had a clear beneficial effect in reducing joint destruction in rheumatoid arthritis [11, 12, 19–21]. From the clinical experience with purified recombinant IL-1Ra it became clear that most of the problems of variability of efficacy were due to difficulties in adequate timing and dosage of the drug . To overcome these problems, gene therapy with adenoviral vectors carrying the IL-1Ra gene has been attempted in experimental animals, yielding promising results in models of type 1 diabetes and ischemic brain damage [22, 23]. The clinical application of the gene therapy approach may however meet with difficulties for safety reasons, besides the problems of controlling drug release, concentration, and localisation.
Based on previous experience of using recombinant bacteria as in vivo cell factory, here we describe a novel system of local delivery of IL-1Ra, able to achieve systemic effects. The system exploits the ability of certain bacteria (such as Bacillus subtilis) to undergo autolysis in non-permissive conditions (as it occurs in the mammalian intestine) thereby releasing the cytoplasmic proteins. Intra-colon instillation of B. subtilis expressing recombinant human mature IL-1Ra induces significant serum levels of the recombinant protein in rats and rabbits, and prevents the inflammatory effects of systemic IL-1. Intra-peritoneal administration of recombinant B. subtilis in the mouse could inhibit LPS-induced shock and death. Further experimental evidence with a B. subtilis strain producing human IL-1β demonstrates that this delivery system can be generalised to other recombinant proteins.
Local and systemic IL-1Ra after administration of IL-1Ra-producing B. subtilis
Delivery of B. subtilis pSM539
Detection of IL-1Ra
+ (B: 149.4 μg)
+/- (B: 16.5 μg)
++ (B: 0.6–1.3 μg/ml)
++ (B: 0.5–1.9 μg/ml)
+ (B: 0.1–0.4 μg/ml)
- (B/E: 0 μg/ml)
+ (B: 0.2–0.4 μg/ml)
++ (B/E: 0.2–1.2 μg/ml)
++ (B/E: 0.6–1.6 μg/ml)
++ (B: 0.6–2.0 μg/ml)
++ (B/E: 0.4–2.1 μg/ml)
++ (B/E: 0.3–0.9 μg/ml)
++ (E: 0.6–2.4 μg/ml)
+ (B: 0.3 μg/ml)
Pharmacokinetic analysis of IL-1Ra in rabbit serum after administration of IL-1Ra-producing B. subtilis
AUC (0–6 h)/dose (g × h)/ml
IL-1Ra + pSM241
The use of live bacteria is very common in particular in vaccinology, where attenuated or mutant bacteria have been employed for decades as antigen carriers. The advantage of live bacteria relies on their capacity of colonising the host and enter the host organs/tissues with the same modalities as their virulent counterparts, thus eliciting the relevant immune response and immune memory, at variance with killed bacteria or purified bacterial components. Thus, attenuated strains of Salmonella, Listeria monocytogenes, Mycobacterium tuberculosis, Vibrio cholerae are being developed and used as vaccine carriers [29–31]. A further development in the use of live bacteria as antigen carriers in vaccination exploits the technologies of genetic engineering for introducing multiple antigens from different micro-organisms into a single non-virulent bacterial carrier (e.g., food-grade lactic acid bacteria), with the possibility of including T- and B-stimulating epitopes from different antigens, and also to engineering into the same carrier adjuvant sequences derived for instance from an immunostimulating cytokine [30–34].
Among bacterial systems developed for antigen delivery in vaccination, some strains of non-pathogenic, food-grade or GRAS (generally regarded as safe) bacteria have been examined for the topical delivery of pharmacologically active protein drugs, after cell engineering with the DNA coding for the protein of interest. This is the case of Lactococcus lactis and of Streptococcus gordonii, which have been engineered to produce recombinant antibodies, adjuvant and anti-inflammatory cytokines, and used to deliver these proteins locally at the mucosal surface after oral administration [3–9]. The goal of these delivery approaches was that of making the recombinant proteins available for therapy of local pathologies or for local effects: antibodies for passive immunotherapy of local infections [3, 4], cytokines as adjuvants for mucosal vaccines [5, 6], inhibitory cytokines for anti-inflammatory therapy of localised chronic inflammatory diseases (IBD-like pathologies) [7–9]. Although undoubtely promising and susceptible of vast applications, the method of mucosal delivery of therapeutic protein through recombinant bacteria acting as cell factories needs further and deeper investigation. This should include the central issue of safety and contained/controlled release of recombinant micro-organisms , the problem of assessing the mucosal permanence of bacteria (extent and duration of colonisation depending on the changes in the mucosal environment in different conditons of health and nutrition) and the extent of protein release, and the issue of pharmacodynamics of the delivered protein in particular for its systemic effects, beyond the boundaries of the local delivery environment.
The delivery system proposed here is not based on the permanence/colonisation capacity of bacteria in the host mucosal surfaces, but it relies on the capacity of sporulating bacteria of releasing intracellular proteins in non-permissive environments. B. subtilis cells engineered to produce human IL-1Ra were able to release the recombinant protein (intact and biologically active) following a sporulation signal in vitro . This observation could be repeated in vivo, when recombinant B. subtilis cells were inoculated in the intestine of rats or rabbits (a non-permissive environment that does not allow the vegetative life of B. subtilis). The recombinant protein could be detected locally shortly after administration of bacteria and persisted at measurable levels for several hours. Release and recovery of recombinant IL-1Ra was much more abundant and consistent in the large intestine as compared to the small intestine. Most interestingly, the recombinant protein released from sporulating bacteria delivered in the large intestine was absorbed in the bloodstream at detectable levels, whereas no circulating IL-1Ra could be found after bacterial delivery in the small intestine. IL-1Ra present in the blood was intact, as judged by its molecular mass in Western blotting, and retained full IL-1-inhibiting activity, as judged by its capacity of dose-dependent neutralisation of IL-1β in vitro. The passage of an intact protein from the intestinal lumen to the bloodstream is not a new concept. Indeed, transcytosis has been extensively described in intestinal epithelial cells, and allows transport of intact proteins and macromolecules from the intestinal lumen to the circulation through an endocytic non-degradative pathway in physiological conditions of integrity of the intestinal mucosal barrier [35–39]. This mechanism of transcytotic transport, quantitatively scarce as compared to the degradative pathway of protein absorption, may have a role in physio-pathological passage of antigens, allergens, and toxins.
Delivery of IL-1Ra through engineered sporulating bacteria apparently had some pharmacokinetics advantages as compared to the purified protein. Whereas the absorption into the bloodstream was quick after administration of the purified protein (Tmax at 60 min), IL-1Ra released from intra-colonically administered B. subtilis had a much slower kinetics of absorption (Tmax 200 min), as expected by the fact that the protein must be released from bacteria before being absorbed. Furthermore, although the Cmax was decreased for B. subtilis IL-1Ra (136 ng/ml vs. 482 ng/ml for the purified protein; only partially attributable to the higher dosage of the purified protein), the AUC/dose were almost identical. Thus, IL-1Ra delivered intra-colonically by B. subtilis is absorbed into the bloodstream at a slower and more constant rate than the purified protein delivered in the same site, which is absorbed quickly into the bloodstream and rapidly disappears thereafter. Thus, it appears that bacteria do not undergo sporulation all at the same time (which would result in a rapidly appearing and disappearing peak of protein), but release the protein constantly from the moment of administration for about 8 h. This would allow a controlled and sustained circulating level of the protein, thus a more favourable pharmacodynamic profile, with a single administration.
The protein selected for in vivo delivery with B. subtilis is the IL-1 receptor antagonist IL-1Ra, a competitive non-activating ligand of the IL-1 receptor with IL-1 inhibitory activity [11, 12]. IL-1 is a potent inflammatory cytokine which, in pathological conditions, is responsible of chronicisation of inflammation, tissue destruction, organ failure, hypotensive shock . Anti-IL-1 strategies have been attempted in acute an chronic inflammatory diseases with the use of recombinant IL-1Ra protein [11, 12]. The poor outcome of clinical trials in septic shock has highlighted the problems of a therapy based on the systemic administration of a purified recombinant protein, whose efficacy is hampered by its rapid pharmacokinetics [11, 12, 18]. At present, experimentation of therapeutic IL-1Ra is being targeted to slowly progressive chronic diseases with defined organ/tissue targets (e.g., rheumatoid arthritis) [40, 41]. To achieve sustained IL-1Ra levels, gene therapy approaches have been attempted with promising results in animal models of experimental arthritis, ischemic brain damage, autoimmune diabetes [19–23]. However, the risk remains of side effects due to the uncontrolled inhibition of the physiologically important IL-1 activity. Indeed, a precise balance between between IL-1β and IL-1Ra should be maintained for achieving proper tissue homeostasis, as shown for the intestinal mucosa .
The drug delivery strategy here described merges the well-known approach of vaccination with live bacteria with that of gene therapy. The delivery of pharmacologically active proteins by live sporulating bacteria, as described here, presents a series of advantages over other similar approaches. At variance with conventional gene therapy, the gene coding for the drug protein is introduced in a bacterial carrier rather than in host cells, a situation that would allow a complete control of its permanence in the body. In a previous study, intragastric or vaginal administration of Streptococcus gordonii engineered to release human IL-1Ra resulted in a prolonged local delivery of the protein, consequent to the capacity of S. gordonii to colonise the mucosal surfaces . Mucosal delivery of IL-1Ra (by intragastric administration of engineered S. gordonii) also had a local therapeutic effect in a model of ulcerative colitis . The delivery system with sporulating bacteria described here differs from that with S. gordonii, as it causes rapid local release of the recombinant protein (e.g. in the large intestine, where IL-1Ra peaks at 4 h and decreases towards background at 24 h), followed by absorption into the bloodstream. In preliminary experiments in the mouse, IL-1Ra-expressing bacteria were also administered intragastrically or subcutaneously. This achieved appearance of human IL-1Ra in the serum, and systemic effects of inhibition of LPS-induced shock and death (data not shown). This is a new finding, that opens the possibility of exploiting localised bacterial administration (e.g. at mucosal sites) for systemic drug delivery. The amount of protein released at the mucosal site directly correlates with the number of administered bacteria, since the internal body environment does not sustain bacterial replication but induces sporulation. This allows an exact control of the dose of drug delivered and, based on the pharmacokinetics parameters, of the blood levels that can be reached. The same result could not be easily obtained with S. gordonii, as amount and timing of protein release may be influenced by variation of the colonisation capacity depending on variations of environmental conditions of the host tissues.
A problem that should be faced when using recombinant bacteria in vivo for therapy or vaccination is that of safety and contained release of genetically modified organisms (GMO). The use of suicidal genes or the deletion of genes vital for survival outside the host organism have been explored with very promising results [8, 43]. The bacterial system proposed here can be modified in the sporulation mechanism for the control of its survival. In preliminary experiments, the recombinant B. subtilis pSM539 strain was engineered in order to inactivate a gene involved in sporulation control. As a consequence, in response to in vitro sporulation signals (adverse environmental conditions) the mutated Spo- strain could regularly initiate the sporulation process, undergoing cell autolysis and release of the cytoplasmic proteins (including the recombinant IL-1Ra), but it was incapable of eventual spore formation and further survival. Likewise, release of the recombinant protein from Spo- in vivo was comparable to that of Spo+ bacteria, but spores could never be recovered from intestinal lavage and faeces (data not shown). This suggests that the system can be optimised to full biological containment and environmental safety without altering its delivery properties.
The novel system of protein drug delivery here proposed links some of the advantages of gene therapy (endogenous production of the relevant protein, targeted delivery) to the possibility of controlled release in terms of timing and protein amount. Exploitation of the mechanism of bacterial autolysis in non-permissive environments allows release of intracellular proteins, including the known amount of the pharmacologically active recombinant protein drug. The release is persistent for several hours, allowing to maintain more constant protein levels in the bloodstream. The system is simple, cheap, and can be developed to full environmental safety (i.e., avoiding the risk of release of genetically modified bacteria in the environment).
The concept that pharmacologically active proteins released at the colonic mucosal surface can be absorbed and reach the circulation intact and retaining full activity (validated with two proteins with opposite effects, IL-1Ra and IL-1β) opens promising avenues to the use of local delivery for the therapy of systemic diseases.
Engineered B. subtilis strains were constructed as previously described in detail [44, 45]. Briefly, cDNA coding for mature human IL-1Ra (encompassing the mutation N91>R), and cDNA coding for mature human IL-1β were cloned between EcoRI and HindIII in pSM214, a B. subtilis plasmid which promotes the synthesis of recombinant products intracellularly, to obtain recombinant plasmids pSM539 (carrying the cDNA for IL-1Ra) and pSM261 (carrying the cDNA for IL-1β). Plasmids were used to transform the B. subtilis SMS118 strain. The pSM539-harbouring B. subtilis strain SMS118(pSM539) could produce 1.0–2.0 mg IL-1Ra/109 cells/0.35–0.49 g (wet weight), after conventional culture overnight in 1 liter flasks. The SMS118(pSM261) strain in the same culture conditions produced 0.15–0.25 mg IL-1β/109 cells/0.35–0.49 g. As negative control, B. subtilis strain SMS118 was transformed with the pSM214 plasmid, which contains the gene of β-lactamase (conferring resistance to penicillin). All strains were leu-, pyrDI, npr-, apr-. Sporulation-defective (Spo-) strains were constructed by mutation in the srfA gene, as previously described  and were kindly provided by Dr. G. Grandi (Chiron S.r.l., Siena, Italy).
Bacteria were grown in LB medium containing 5 mg/l chloramphenicol for 7 h at 37°C under shaking and harvested by centrifugation (3,000 × g, 20 min, 4°C). For sporulation supernatant preparation, 0.25 g wet weight of bacteria (corresponding to 0.5–0.7 × 109 cells) were suspended in Difco sporulating medium (bacto beef extract 3 g/l, peptone 5 g/l, NaOH 0.25 mM, MgSO4 10 mM, KCl 0.1%, MnCl2 0.1 mM, Ca(NO3)2 1 mM, FeSO4 1 mM, pH 6.8) without chloramphenicol and incubated at 35°C with shaking. Aliquots of sporulation supernatant were harvested by centrifugation (14,000 × g, 5 min) at different time points. Following sporulation signals, both Spo+ and Spo- bacteria initiate the autolysis process, which ends in cell autolysis with release of cytoplasmic content. However, whereas in Spo+ bacteria there is formation of a spore with preservation of strain survival, Spo- bacteria are unable to form a spore thus undergoing complete cell destruction (Figure 1A). Upon sporulation signals, both Spo+ and Spo- bacteria released 100% of intracellular recombinant products in a time-dependent fashion, with maximal relaease between 2 an 8 h (Figure 1C) .
Protein samples were run on 13.5% mini SDS-PAGE according to Lämmli  and stained with Coomassie R-250. The gel was subjected to laser scanning on a Molecular Dynamics Personal Densitometer, and the densitometric analysis was made using Image Quant software (Molecular Dynamics, Sunnyvale, CA).
Experimental animals were: female C3H/HeOuJ mice of 10–12 weeks of age (20–25 g) (for all in vivo experiments), female C3H/HeJ mice of 5–8 weeks of age (for thymocyte proliferation), female Sprague-Dawley rats (around 300 g), and female New Zealand rabbits (1.9–2.5 kg). All animals were purchased from Charles River Italia (Calco, Italy) and were housed in standard cages at 22 ± 1°C with 12 h light-12 h dark cycle. Animals received standard diet and tap water ad libitum.
In vivo administration of B. subtilis
Bacteria were harvested and resuspended in LB medium or sterile PBS.
Mice received a single intra-peritoneal injection of 0.2 ml of bacterial suspension in PBS.
Rats were fasted overnight before the surgical procedure and maintained under urethane anaesthesia throughout. Bacteria (in LB medium diluted 1:1 in PBS) were instilled in the small intestine (duodenum) with a 22 1/2 G needle, in a volume of 1–10 ml. Two surgical ligatures were applied, one at the beginning of the duodenum immediately below the needle entry puncture (to avoid exit of instilled bacteria), and another one near the ileocecal valve, to limit to the small intestine the transit of bacteria. Intra-colon instillation was performed again with a 22 1/2 G needle in the ceacum immediately below the ileo-caecal valve, in a volume of 5–10 ml. Two surgical ligature were applied just below the needle entry point and at the colon terminal region, to avoid loss of bacteria. Animals were sacrificed by exanguination at different times after treatment, to collect blood and intestinal washings.
For intra-colonic administration of bacteria in rabbits, animals were fasted overnight prior to treatment, then lightly restrained in conventional stocks and maintained conscious throughout the experiment. A rounded-tip urethral catheter (Rüsh, Germany) was carefully inserted 10 cm into the distal colon via the anal route and 2 ml of B. subtilis suspension were administered. Serum samples were prepared from blood collected from the rabbit marginal ear vein at different times (0–8 h) after intra-colonic administration of bacteria. In some experiments, animals were sacrificed, to collect the large intestine content (saline washing).
Protocols of animal experimentation were reviewed by the institutional ethical board for adherence to ethical guidelines for animal research conduct (Italian D. L.vo 27/01/1992 n. 116 and corresponding EU directive 86/609; policy of refinement, reduction and replacement towards the use of animals for scientific procedures 99/167/EC – Council Decision of 25/1/99), and previously authorised by the Italian Ministry of Health.
Detection of human IL-1Ra in animal samples
Western blotting: samples were subjected to reducing 15% mini SDS-PAGE and analysed by Western blotting using a polyclonal rabbit serum anti-human IL-1Ra and a goat anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase, as described in detail elsewhere . Serum samples were filtered on Microcon 100 (MWCO 100,000; Amicon, Beverly, MA) before analysis.
ELISA measurement: samples were subjected to quantitative determination of human IL-1Ra using a specific ELISA (Amersham, Little Chalfont, UK), following the manufacturer's instructions. The lower detection limit was 20 pg/ml. Purified human recombinant IL-1Ra was used as standard. Serum samples were filtered on Microcon 100 (Amicon) before analysis.
Biosensor measurement: detection of IL-1Ra in serum samples and intestinal washings was confirmed with the biosensor BIAcore™ system (Pharmacia Biosensor AB, Uppsala, Sweden), which allows real time biospecific interaction analysis by means of the optical phenomenon of surface plasmon resonance, as previously described in detail . The lower detection limit for human IL-1Ra was 2 pg/ml.
IL-1-induced thymocyte proliferation
The classical assay of co-stimulation of murine thymocyte proliferation was used to evaluate the bioactivity of IL-1 and IL-1Ra. Briefly, thymocytes from 5–8 week-old C3H/HeJ mice (preferentially used because of their LPS unresponsiveness) were cultured at 6 × 105 cells/well of Cluster96 plates (Costar, Cambridge, MA) in 0.2 ml of RPMI-1640 medium (Life Technologies, Paisley, Scotland) supplemented with 2 mM L-glutamine, 25 mM HEPES buffer, 50 μg/ml gentamycin sulfate, 1.25 × 10-5 M 2-ME (all from Sigma Chemical Co.), 5% fetal bovine serum (Hyclone, Logan, UT) for 72 h in moist air with 5% CO2 . The biological activity of IL-1β was assessed as co-stimulation of thymocyte proliferation, by adding to the culture wells a selected amount of human recombinant IL-1β (30–300 pg/ml)  and a suboptimal concentration of purified PHA (1.5 μg/ml; Murex Diagnostics, Dartford, UK). Cells were then pulsed for 18 h with 18.5 kBq/well of [3H]TdR (sp. act. 185 GBq/mmol; Amersham) and their proliferation was measured as radiolabel incorporation with a β-counter.
The biological activity of IL-1Ra was evaluated as inhibition of IL-1β-dependent thymocyte proliferation. To this end, cells were stimulated to proliferate (with IL-1β and PHA) in the presence of increasing concentrations of human recombinant IL-1Ra  or serial dilutions of serum from rabbits receiving pSM214 or pSM539 intra-colonically, or from untreated rabbits. The concentration of IL-1Ra in serum of pSM539-treated rabbits was determined by ELISA and serum was added to the cultures after appropriate dilution. Control sera from pSM214-treated or untreated rabbits were used at the same dilutions as IL-1Ra-containing serum. Cell proliferation was then evaluated as radiolabel incorporation as described above.
To assess that the effect of IL-1Ra-containing serum was indeed due to IL-1Ra, a polyclonal rabbit antibody against human IL-1Ra  was added to the cultures at a dilution of 1:300, i.e. the dilution previously found to inhibit 50% of the activity of 10 ng/ml IL-1Ra in the thymocyte assay (not shown).
Rabbits were lightly restrained in conventional stocks throughout the experiment, and accustomed to the stocks over a period of 2 h, to minimise variations in body temperature. Body temperature was measured by means of a cutaneous thermistor probe (TM-54/S and TMN/S; LSI-Lastem, Settala Premenugo, Italy) placed between the left posterior paw and the abdomen and allowed to stabilise for 2 min. B. subtilis suspensions (2 × 109 live cells/rabbit) were instilled in the distal colon 1 h before i.v. administration of 50–100 ng/ml highly purified LPS-free human recombinant IL-1β in pyrogen-free saline through the marginal ear vein. Temperature was recorded every 20 min for 3 h starting from IL-1β administration. In experiments with IL-1β-producing strain pSM261, rabbits received an intra-colonic administration of 1 × 109 pSM214 (control) or pSM261 (IL-1β) bacteria, or 250 μg purified human IL-1β. Temperature was recorded up to 22 h after treatment.
IL-1-induced neutrophilia, hypoferremia, hypoglycemia
Live cells of B. subtilis strains pSM214 and pSM539 were instilled in the distal colon (1 × 109 cells/kg), 2 h before administration of IL-1β. Blood samples were drawn 2, 4, 6, 8 and 24 h after intra-peritoneal inoculum of 0.1 μg/kg human recombinant IL-1β. The number of circulating neutrophils was assessed by flow cytometry. The plasma iron concentration was determined colorimetrically with a commercially available kit (Fe; Boehringer Mannheim, Mannheim, Germany). Hypoferremia (60–75% decrease of plasma iron level) was evident from 4 to 24 h after IL-1β inoculum. The blood glucose concentration was measured in serum samples by the glucose/glucose oxidase/peroxidase method with commercially available kits (Glucose GOD Perid; Boehringer Mannheim) or by biosensor detection with devices for diagnostic monitoring (Roche Diagnostics, Milano, Italy). Overlapping results were obtained in rats and rabbits.
LPS-induced shock in the mouse
LPS-sensitive C3H/HeOuJ mice received an intra-peritoneal inoculum of 0.5 ml PBS alone or containing bacterial suspensions (control pSM214, IL-1Ra-producing pSM539; 3 × 106 bacteria/mouse), 24 h before i.p. administration of 15–20 mg/kg of LPS (from E. coli 055:B5; Sigma Chemical Co., St. Louis, MO). LPS inoculum was delayed to 24 h after bacteria administration to avoid interference of pre-inoculum. In fact, preliminary experiments showed that intra-peritoneal inoculum of PBS decreased significantly LPS toxicity when administered at shorter times before LPS (data not shown). Mice were observed for 7 days after LPS administration and deaths recorded.
Results are presented as mean ± SEM. Statistical significance was assesed by two-tailed Student's t test. Comparison of survival curves was performed by the χ2 test. Calculation of percentiles was performed by survival analysis. All calculations were performed with the Stratgraphics Plus 5 programme (Manugistics, Inc., Rockville, MD).
List of abbreviations
interleukin-1 receptor antagonist
inflammatory bowel disease
bacterial lipopolysaccharide, AUC, area under the curve
generally regarded as safe
genetically modified organisms.
This work was supported by the Commission of the European Union (contract no. QLK4-2001-00147), a research grant from AIRC (Associazione Italiana Ricerca sul Cancro), Milano, Italy, and the FIRB project "NIRAM" of the Italian MIUR.
The authors are particularly indebted to Giovanni Maurizi (Consorzio Biolaq, L'Aquila, Italy) for his seminal contribution to this work. The support of Cinzia D'Ettorre (Dompé SpA, L'Aquila, Italy) for BIAcore analysis is gratefully acknowledged.
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