WO2009093900A1 - Mid-log phase lactic acid bacteria for inducing immune tolerance in a subject - Google Patents

Abstract

The invention relates to a composition comprising lactic acid bacteria, wherein a substantial amount thereof is in the mid-log phase, to its use for inducing immune tolerance and to a device for administering it.

Description

P6018151 Mid-log phase lactic acid bacteria for inducing immune tolerance in a subject

Field of the invention The invention relates to a composition comprising lactic acid bacteria, wherein a substantial amount thereof is in the mid-log phase, to its use for inducing immune tolerance and to a device for administering it.

Background of the invention Human intestinal epithelia are continuously exposed to the resident microbiota that is present in the gut lumen. In healthy humans, this continuous exposure to non-self stimuli is now known to activate tightly controlled, and inter-connected, innate and adaptive immune responses (Medzhitov and Janeway Jr., 2002; Clavel and Haller, 2007; Kabelitz and Medzhitov, 2007; McHeyzer-Williams, 2007; MacPherson and McCoy, 2007) including the production of antimicrobial factors (Dann and Eckmann, 2007). In addition to the resident bacterial load, nonpathogenic and potentially pathogenic bacteria are ingested via the food on a daily basis. In order to avoid disease, the gut epithelia continously monitor resident and ingested, non-resident or transient bacteria. Epithelia have to respond differentially to antigens and adjuvants from pathogenic and non-pathogenic bacteria to avoid overly aggressive, inappropriate or disproportionate inflammatory responses that may lead to tissue damage, a feature of Crohn's disease (Balfour Sartor, 2007; Baumgart and Carding, 2007). In healthy adult humans, controlled and balanced immune responses lead to tolerance towards commensal bacteria. Appropriate inflammatory responses to pathogenic bacteria and strictly contained and modulated or "dampened" innate immune responses to nonpathogenic microbiota is dependent on activation and termination of correlating immune responses, together achieving immune homeostasis (Cario and Podolsky, 2000; Sansonetti, 2006; Sansonetti and di Santo, 2007).

Intestinal epithelial cells (IECs) play a crucial role in mediating immune responses. For instance, the initiation and modulation of appropriate immune responses in response to microbial stimuli and downstream processes such as class switching of mucosal protective immunoglobulin A (IgA) and dendritic cell (DC)-mediated stimulation of naive B cells to produce protective antibodies are important features of IECs (He et al., 2007; McHeyzer- Williams, 2007; Xu et al., 2007). The innate immune response towards microbes is composed of a signalling network passing on signals from receptors that identify bacteria or their products. Commensal bacteria induce transient, mainly non-inflammatory host responses, whereas pathogens continuously induce the production and release of pro-inflammatory responses both directly and indirectly, through their capacity to damage cells via toxins or by altering host signalling pathways through secreted factors (Foster, 2005; Sansonetti and di Santo, 2007; Schnaith et al., 2007). For instance, the induction of non- inflammatory immune responses by nonpathogenic Lactobacillus sp. can be accomplished via secreted factors (e.g. as recently found for L. rhamnosum; Yan et al., 2007) or via wall antigens or adjuvants as found for L. plantarum, a well-studied nonpathogenic and versatile food- grade bacterial species with a completely sequenced genome (Kleerebezem et al., 2003).

L. plantarum has been the subject of diverse immunological investigations. This bacterial species was isolated from a mistletoe preparation (called Iscador®) with known adjuvanticity (Bloksma et al., 1979a,b). L. plantarum was eventually isolated as the main bacterial species underlying the adjuvant capacity of the mistletoe extracts in an experimental mouse model system (Bloksma et al., 1979b). Iscador® has been shown to promote the production of immunoglobulin (Ig)-M, IgG and IgA antibodies upon administration to mice (Bloksma et al., 1979a). More recently, Iscador® was also shown to induce IgG antibody production by human mononuclear cells; in addition, it also induced production of the cytokines TNF-α and IL-12, partially mediated through the CD14 receptor (Heinzerling et al., 2006). Strikingly, upon stimulation by L. plantarum, one of the major bacterial species isolated from Iscador®, human monocytes were also shown to produce IL-12 and TNF-α (Karlsson et al., 2004). L. plantarum can interact with, or signal through TLR2, TLR4 and the CD 14 receptor (Karlsson et al., 2002, 2004). It is therefore possible that the reported adjuvanticity of Iscador® is mainly caused by L. plantarum. In addition to stimulation of the extracellular TLR2, TLR4 and CD 14 receptor, L. plantarum also stimulates the intracellular receptor Nod2 (Hasegawa et al., 2006).

L. plantarum adjuvanticity exerts distinct effects in a mouse experimental model depending on the viability of bacterial preparations. Viable bacteria induce delayed (48-72 hrs) mild inflammatory responses, executed via mononuclear leukocytes (delayed hypersensitivity) when administered between 8h before up to 24 hrs after a second immuno-stimulant. In contrast, dead bacteria administered 8h before or together with an immuno-stimulant induce antibody production and presentation (Bloksma et al, 1979b). Subcutaneous injection of mice with live 10^s L. plantarum resulted into development of mild, granulomatous tissue inflammation and infiltration of immune cells into the connective tissue within 3 days. Viable L. plantarum induced the infiltration of mainly mononuclear cells into the tissue, whereas heat-killed L. plantarum induced an infiltration of about equal numbers of granulocytes and mononuclear cells (Bloksma et al., 1979b). The delayed hypersensitivity and antibody production were considered to be brought about via different cellular mechanisms, showing that modulation of immune responses by Lactobacillus species may be exerted via more than one mechanism. More recently, it was shown that Lactobacillus can promote surface expression of several receptors (including CD40 and CD86) in immature human myeloid dendritic cells (DCs) and induce secretion of IL-12 and IL- 18 in DCs, leading to a polarisation of CD4+/CD8+ T cells towards regulatory T cells (Mohamadzadeh et al., 2005), again highlighting one complex way by which Lactobacillus species can modulate (adaptive) immune responses.

Nowadays lactic acid bacteria are broadly consumed and are believed to be involved in potentially several distinct immunomodulating properties on a subject being administered the lactic acid bacteria (adjuvant effect, pro-inflammatory, antiinflammatory effect, appropriate balance between innate and adaptive immune responses through stimulation of regulatory T cell maturation). However, as illustrated above, the mechanisms underlying these several distinct effects on the immune system are far from being elucidated and comprised. Especially, it would be attractive to get a composition comprising lactic acid bacteria, wherein this composition would have one single defined specific effect on the immune system of a subject being administered the composition. There is a need for an improved composition comprising lactic acid bacteria, wherein this composition would have as main effect a tolerance-promoting effect on the immune system.

Description of the invention

The present inventors unraveled at least part of the mechanisms potentially explaining when and how a tolerance-promoting effect may be exerced on the immune system by lactic acid bacteria. Surprisingly, they discovered that an important parameter for inducive immune tolerance is linked to the growth stage of the lactic acid bacteria used as explained below.

So far, the effect of Lactobacillus species on modulation of immune responses has been studied in animal models or human cell lines. To start investigating in vivo responses of human to Lactobacillus, we chose to expose healthy human volunteers to L. plantarum since this bacterial species has already been well studied and is known to possess potent adjuvanticity (see earlier). Transcriptional profiling was employed in order to analyse the host response since, during the last decade, global transcriptional profiling of human cell lines mounting immune responses to pathogenic bacteria has demonstrated to be very useful (Baldwin et al, 2002; Nau et al, 2002; Kobayashi et al, 2003; McCaffrey et al., 2004; Rinella et al., 2006; Eickhoff et al., 2007). However, all of these in vitro studies still employed highly simplified cell line models. Therefore, it remained to be established whether these provide an appropriate reflection of the complex in vivo situation, stressing the lack of in vivo data for interactions of human with pathogenic and non-pathogenic bacteria. We chose to perform a comprehensive in vivo analysis of epithelial responses of the duodenal part of the small intestine of adults after prolonged (six hours) exposure to L. plantarum. In order to get a genome-wide overview of cellular responses to L. plantarum, we obtained transcriptional profiles that were altered as a result of the in vivo interaction of epithelia with L. plantarum. The volunteers were exposed to actively growing and metabolising midlogarithmic-phase- bacteria (exponentially growing; we will refer to this preparation as "midlog") as well as to quiescent, stationary-phase-grown (or "stationary") bacteria. We also included a trial using heat-killed ("dead") L. plantarum to assess whether dead or living bacteria would induce differential responses in human, as shown in mouse (Bloksma et al., 1979b). Our clinical trial, set up according to a double-blind placebo-controlled crossover design, provides a unique data set of human responses to ubiquitous food-grade bacteria. The expression profiles of healthy adults during interaction with L. plantarum uncovered in this study reflect the establishment of immune tolerance towards nonpathogenic bacteria in the duodenal part of the small intestine. Composition

In a first aspect, there is provided a composition comprising lactic acid bacteria, wherein a substantial amount of the lactic acid bacteria is in the mid-log phase. The skilled person will understand that a lactic acid bacteria used in a composition of the invention could be distinct from the herein specified Lactobacillus plantarum strain, eg, from other Lacobacilli species or even from other lactic acid bacteria probiotic species as long as it has the identity and functionality both as herein defined. Preferred lactic acid bacterium belongs to a genus selected from the group consisting of Lactobacillus, Lactococcus, Leuconostoc, Carnobacterium, Streptococcus, Bifidobacterium, Bacteroides, Eubacterium, Clostridium, Fusobacterium, Propionibacterium, Enterococcus, Staphylococcus, Peptostreptococcus, and Escherichia. A further preferred lactic acid bacterium is a Lactobacillus or Bifidobacterium species selected from the group consisting of L. reuteri, L. fermentum, L. acidophilus, L. crispatus, L. gasseri, L. johnsonii, L. plantarum, L. paracasei, L. murinus, L.jensenii, L. salivarius, L. minutis, L. brevis, L. gallinarum, L. amylovorus, B. bifidum, B. longum, B. infantis, B. breve, B. adolescente, B. animalis, B. gallinarum, B. magnum, and B. thermophilum. Preferably, a lactic acid bacteria used is not one of Lactococcus lactis diacetilactis or Streptococcus diacetilactis.

Accordingly in a preferred embodiment, a lactic acid bacterium used is a Lactobacillus plantarum strain. More preferably the Lactobacillus plantarum strain used is WCFSl. The strain WCFSl is a single colony isolate from L. plantarum NCIMB8826, which was originally isolated from human saliva. The strain is present in the National Collection of Industrial and Marine Bacteria, Aberdeen, U.K. (ES). This strain has been sequenced (Kleerebezem M.,et al. (2003), Proc. Natl. Acad. ScL, vol 100: 1990-1995).

Within the context of the invention, "a substantial amount" preferably means at least 50% of the total lactic acid bacteria number of cells, more preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more. Most preferably, "substantial amount" means all lactic acid bacteria. Cells are preferably counted under the microscope. A composition of the invention may therefore comprise an amount of lactic acid bacteria ranged between 10³ and approximately 10¹³ cells that are present in the midlog phase, preferably between 10³ and 10⁶. Within the context of the invention, "mid-log phase" preferably means actively growing or exponentially growing or metabolising mid-logarithmic-phase bacteria or mid-logarithmic-phase bacteria. It means that mid-log phase bacteria are in the exponential phase of their growth. As the skilled person knows per definition during the exponential phase, a 2-log scale or a 10-log scale provides a linear relation between the density of cells and the time. Mid- log phase bacteria is opposed to late log or to quiescent stationary phase grown bacteria. In a preferred embodiment, when lactic acid bacteria are cultured in a standard classical such as medium MRS under non-aerobic or micro-aerophile conditions at 30⁰C starting with a 1:1000 inoculum size from an overnight grown culture, mid-log phase bacteria are obtained after approximately 2 to approximately 6 hours of culture. The skilled person knows how to assess whether bacteria are mid-log phase bacteria by counting cells regularly and/or by measuring the optical density at 600 nm. Bacteria are in mid-log phase when their culture density increases logarithmically over time. In most bacterial cultures and with most culture media, this period starts shortly after the moment growth initiates (first signs of growth measured as an increase of density relative to the inoculum density) until the rate of growth starts to decrease and cells enter the transition stage to stationary phase of growth (recognizable as a reduction of growth rate as compared to the logarithmic growth phase) (Growth measurement, 1981, Koch A.L., Chapter 11 in "Manual of Methods for General Bacteriology" Editors: Gerhardt, P., Murray, R.G.E., Costilow, R.N., Nester, E. W., Wood, W.A., Krieg, N. R., and Briggs Phillips, G., American Society for Microbiology, ASM-Press, Washington D. C, 1981).

To prepare a composition as defined herein, a lactic acid bacteria is cultured under appropriate conditions, optionally recovered from the culture medium and optionally formulated into a composition suitable for the intended use. It is crucial the growth phase of the lactic acid bacteria is assessed in order to get a composition as defined above. Methods for the preparation of such compositions are known per se. Compositions for enteral or oral administration may be either food compositions or pharmaceutical compositions whereas compositions for nasal, vaginal or rectal administration will usually be pharmaceutical compositions. A pharmaceutical composition will usually comprise a pharmaceutical carrier in addition to the host cells of the invention (i.e. lactic acid bacteria). The preferred form depends on the intended mode of administration and (therapeutic) application. A pharmaceutical carrier can be any compatible, nontoxic substance suitable to deliver the host cells of the invention to the GI-tract of a subject. E.g. sterile water, or inert solids may be used as the carrier usually complemented with pharmaceutically acceptable adjuvants, buffering agents, dispersing agents, and the like. A compositions will either be in liquid, e.g. a stabilized suspension of the host cells, or in solid and/or dry forms, e.g. a powder of lyophilized host cells. E.g. for oral administration, the host cells can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions.

Alternatively or in combination with other preferred embodiments mentioned herein, the invention discloses another preferred embodiment, a composition of the invention comprising alive and/or activatable lactic acid bacteria. Within the context of the invention, "alive" is understood to mean not dead. Preferably, "alive" means that a lactic acid bacteria is able to grow as a midlog lactic acid bacteria under normal culture conditions known to the skilled person (MRS medium, under non-aerobic or micro-aerophile conditions at 30⁰C starting with a 1:1000 inoculum size from an overnight grown culture) . The ability to grow is preferably assessed by measuring the optical density of the culture at 600 nm. A detectable increase in OD_OOO is considered to indicate that a lactic acid bacterium is alive. Alternatively, if cells transferred from the liquid culture onto plates comprising a solid medium are able to form colonies, then these cells are considered to be alive. Within the context of the invention, "activatable" preferably means that upon contact with a moist, the lactic acid bacteria as defined herein will be activated, i.e. awaked and will continue their mid-log growth and exert their desired specific effect on the immune system of a subject treated. Such activation may be obtained when a subject in a need of a lactic acid bacteria as defined herein will ingest a dry form thereof and/or when a subject will use a device according to the invention as later on defined herein. A dry form of a lactic acid bacteria is defined as being an alive and activatable form of a lactic acid bacteria.

Alternatively or in combination with other preferred embodiments mentioned herein, the invention discloses another preferred embodiment, a composition of the invention comprising a dry form of a lactic acid bacteria. This embodiment is highly preferred since it allows to isolate, enrich and store a lactic acid bacteria having reached a midlog phase as defined herein. Within the context of the invention, "dry form" preferably means that a lactic acid bacterium is in a dry form including but not limited to a powder, a granulate, and/or a solid. "Dry" preferably means that the total moisture content of the bacteria is less than about 10 weight %, more preferably less than about 5 weight % and most preferably less than about 1%. The bacteria is preferably freeze- dried or lyophilised, although any method for drying the bacteria may be used. The host cells of the invention may be dried and later on mixed with an inactive ingredient and/or excipient and optionally encapsulated with for example gelatin to form gelatine capsules. Alternatively, the host cell may be tabletted. Inactive ingredients are such as flavoring agents, stabilizers, sugars or other energy sources, buffering agents, thickeners, diluents, dispersing aids, emulsifiers, and/or binders. Examples of inactive agents are: glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like. The host cells may also be first added to the bacteria in a liquid form, after which the combination is dried. The use of a carbohydrate enriched media such as a dairy product, preferably milk may be used to this end. In a more preferred embodiment, a composition comprises a dry activatable form of a lactic acid bacteria as defined herein.

A preferred composition according to the invention is suitable for consumption by a subject, preferably a human or an animal. Such compositions may be in the form of a food supplement or a food or food composition, which besides the host cells of the invention also contains a suitable food base. A food or food composition is herein understood to include liquids for human or animal consumption, i.e. a drink or beverage. The food or food composition may be a solid, semi-solid and/or liquid food or food composition, and in particular may be a dairy product, such as a fermented dairy product, including but not limited to a yoghurt, a yoghurt-based drink or buttermilk. In a preferred embodiment, a food supplement is intended for use in an infant formulae. In another preferred embodiment, a food composition is an infant formulae. Such foods or food compositions may be prepared in a manner known per se, e.g. by adding the host cells of the invention to a suitable food or food base, in a suitable amount. In a further preferred embodiment, the host cells are micro-organisms that are used in or for the preparation of a food or food composition, e.g. by fermentation. Examples of such micro-organisms include lactic acid bacteria, such as probiotic lactic acid strains as earlier exemplified herein. In doing so, the host cells of the invention may be used in a manner known per se for the preparation of such fermented foods or food compositions, e.g. in a manner known per se for the preparation of fermented dairy products using lactic acid bacteria. In such methods, the host cells of the invention may be used in addition to the micro-organism usually used, and/or may replace one or more or part of the micro-organism usually used. For example, in the preparation of fermented dairy products such as yoghurt or yoghurt- based drinks, a food grade lactic acid bacterium of the invention may be added to or used as part of a starter culture or may be suitably added during such a fermentation or may be added at the end of a fermentation. Preferably, a lactic acid bacterium is added at the end of the fermentation. Preferably, a composition as defined above will contain a host cell of the invention in amounts that allow for convenient (oral) administration of the host cells of the invention, e.g. as or in one or more doses per day or per week. In particular, a preparation or composition may contain a unit dose of the host cells of the invention. A composition of the invention may comprise an amount of lactic acid bacteria ranged between approximately 10⁷ and approximately 10¹³. Preferably, approximately 10⁹ and approximately 10¹¹. A composition of the invention may comprise an amount of lactic acid bacteria ranged between 10⁷ and 10¹³. Preferably, 10⁹ and 10¹¹.

Alternatively or in combination with other preferred embodiments mentioned herein, the invention discloses in another preferred embodiment, a composition as defined herein, which is capable of inducing (down- or up-regulating) a gene expression profile indicative of immune tolerance when administrated to a subject. A preferred gene whose expression is down-regulated by a composition of the invention is the TREMl (Triggering Receptor Expressed on Myeloid cells 1) gene, whose gene product is a receptor that regulates and amplifies pro-inflammatory signals) and epithelial layer growth (e.g. the genes MYC and cyclin Dl ). A preferred gene whose expression is up- regulated is the important proliferation- and DNA metabolism-associated PARPl (poly (ADP-ribose) polymerase family, member 1) gene. This gene is specifically up- regulated during the response to midlog L. plantarum. In a more preferred embodiment, a composition as defined herein is provided, which is capable of down-regulating the expression of a TREMl gene and/or up-regulating the expression of a PARPl gene when administered to a subject.

Alternatively or in combination with earlier preferred embodiments mentioned herein, the invention discloses in another preferred embodiment, a composition as defined herein which is further characterised by the substantially non-induction and/or non expression of immune response-related genes when administrated to a subject. Immune response-related genes are preferably selected among the following genes: CXCL2, JNK, NF-Kb subunits p50, plOO and ReIB.

An important cytokine that is not regulated in response to midlog L. plantarum but regulated in response to stationary and heat-killed L. plantarum is CXCL2 (chemokine (C-X-C motif) ligand 2). The immune response-promoting kinase JNK (Janus N- terminal kinase, syn. with MAPK8, mitogen-activated protein kinase 8) and the NF-κB (nuclear factor KB) subunits p50 (NFKB l), p i 00 (NFKB2) and ReIB (v-rel reticuloendotheliosis viral oncogene homolog B) are also not regulated in response to midlog L. plantarum.

Regulation of NF-κB, including the up-regulated expression of the NF-κB modulator BCL-3, is an important prerequisite for any tissue to correctly distinguish self from non-self. Being able to make this distinction is one major aspect of immune tolerance (Cario and Podolsky, 2005; Zhang et al, 2007).

Each of the genes as defined herein is identified by a specific nucleotide sequence coding for a corresponding protein also as defined herein. Each of TREMl, PARPl, CXCL2, JNK, NF-Kb subunits p50, p i 00 or ReIB is identified by a nucleotide sequence encoding said named protein and having at least 60% identity with the nucleic acid sequence SEQ ID NO:1, 2, 3, 4, 5, 6, or 7 respectively as identified in Table 8. Alternatively, TREMl, JNK, NF-Kb subunits p50, pi 00, ReIB, PARPl or CXCL2 is each identified by an amino acid sequence representing said named protein and having at least 60% identity with the amino acid sequence SEQ ID NO:8, 9, 10, 11,12, 23 or 24 respectively as identified in Table 8. More preferably, the identity percentage is of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100%. The assessment of the expression level of a nucleotide sequence or a gene as identified herein is preferably performed using classical molecular biology techniques such as (real time) PCR, arrays or Northern analysis. Alternatively, according to another preferred embodiment, the expression level of a nucleotide sequence is determined indirectly by quantifying the amount of a polypeptide encoded by said nucleotide sequence. Quantifying a polypeptide amount may be carried out by any known techniques. Preferably, polypeptide amount is quantified by Western blotting. The skilled person will understand that alternatively or in combination with the quantification of an identified nucleic acid sequence and/or corresponding polypeptide, the quantification of a substrate of a corresponding polypeptide or of any compound known to be associated with the function of said corresponding polypeptide or the quantification of the function or activity of said corresponding polypeptide using a specific assay is further encompassed within the scope of the invention. Since the expression level of a nucleotide sequence and/or amounts of a corresponding polypeptide may be difficult to be measured in a subject, a sample from a subject is preferably used. According to another preferred embodiment, the expression level (of a nucleotide sequence or polypeptide) is determined ex vivo in a sample obtained from a subject. The sample preferably comprises an intestinal sample (e.g. a biopsy) from a subject. Biopsies (mucosal tissue samples) from the horizontal part of the duodenum can be obtained by standard flexible gastroduodenoscopy in most subjects.

An up-regulation or down-regulation of the expression level of a nucleotide sequence (or steady state level of the encoded polypeptide) is preferably defined as being a detectable change of the expression level of a nucleotide (or steady state level of said encoded polypeptide or any detectable change in the biological activity of said polypeptide) using a method as defined earlier on as compared to the expression level of said corresponding nucleotide sequence (or steady state level of said corresponding encoded polypeptide) in the subject before administration of a composition of the invention or in a subject which has not been administrated a composition of the invention. According to a preferred embodiment, an up-regulation or down-regulation of a polypeptide activity is quantified using a specific assay for the polypeptide activity. Depending on the polypeptide, the skilled person will know which assay is the most suited. Preferably, an up-regulation or increase of the expression level of a nucleotide sequence or gene means an increase of at least 5% of the expression level of a nucleotide sequence using micro-arrays. More preferably, an increase of the expression level of a nucleotide sequence means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150% or more.

Preferably, a decrease or down-regulation of the expression level of a nucleotide sequence or gene means a decrease of at least 5% of the expression level of said nucleotide sequence using micro-arrays. More preferably, a decrease of the expression level of a nucleotide sequence means an decrease of at least 10%, even more preferably at least 20%., at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150% or more.

Preferably, an increase or up-regulation of the expression level of a polypeptide means an increase of at least 5% of the expression level of said polypeptide using western blotting. More preferably, an increase of the expression level of a polypeptide means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150% or more.

Preferably, a decrease or down-regulation of the expression level of a polypeptide means a decrease of at least 5% of the expression level of said polypeptide using western blotting. More preferably, a decrease of the expression level of a polypeptide means a decrease of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150% or more.

Preferably, an increase or up-regulation of a polypeptide activity means an increase of at least 5% of said polypeptide activity using a suitable assay. More preferably, an increase of a polypeptide activity means an increase of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least

70%, at least 90%, at least 150% or more.

Preferably, a decrease or down-regulation of a polypeptide activity means a decrease of at least 5% of said polypeptide activity using a suitable assay. More preferably, a decrease of a polypeptide activity means a decrease of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, at least 150% or more. Preferably, the substantially non- induction and/or non expression of an immune response-related gene as identified earlier herein means that no increase and no decrease of at least one of such genes or polypeptide is detectable using any of the methods as identified herein. Increase or decrease is in this context given the same meaning as earlier identified herein.

Tolerance induction may have a major impact on a variety of aberrations of the immune system: for example allergic diseases may be treated and/or prevented and/or progression of allergic diseases may be delayed by increasing tolerance of the intestinal and/or systemic immune system. Treatment of allergy preferably means allergy symptom reduction. Prevention of allergy preferably means reduction of risk of allergy development.

In a further preferred embodiment, a composition of the invention does not modulate or regulate Microsomal Triglyceride Transfer Protein (MTP) gene expression. Alternatively or in combination with other preferred embodiments mentioned herein, the invention discloses another preferred embodiment, which is a composition or a device as defined herein is for use as a medicament. Such composition or device, is for preventing, delaying and/or treating at least one of a condition or disease associated with an aggressive, inappropriate, and/or disproportionate inflammatory response that can lead to tissue damage. Preferred conditions or diseases are such as allergy, autoimmune disease, or inflammatory disorders of the intestine as already identified herein.

Device In a further aspect, there is provided a device for administering a dry composition as defined in the previous section, comprising a first compartment for holding a moist component, a second compartment for holding this dry composition and a separator for separating said first and second compartment, such that when said separator is at least partially removed, said moist component and said dry composition are permitted to mix to form a mixture, thereby forming a composition as earlier defined herein. This type of device has been extensively described in WO 05/034861.

This device is quite attractive since a lactic acid bacteria in its dry form is kept in its mid-log status as defined earlier on. Upon removing a separator, a lactic acid bacterium is specifically activated by being moistened by the content of the second compartment before being administered to a subject in a need thereof.

A moist component of the first compartment may be any edible moisture. It may be a liquid medium such as an aqueous medium. A moist component may alternatively be a semi-solid formulation such as a gel, a paste, a pudding or a yoghurt.

The removal (or detachment) of a separator may be total or partial. Partial removal preferably means pierced.

Use Another aspect of the invention relates to the use of a composition or a device according to the invention in the preparation of a medicament for preventing, delaying and/or treating a disease or a condition associated with an aggressive, inappropriate, and/or disproportionate inflammatory response that can lead to tissue damage. Preferred conditions or diseases are such as allergy, autoimmune disease, or inflammatory disorders of the intestine as already identified herein.

Method

Another aspect of the invention relates to a method wherein a composition or a device according to the invention is administered to a subject in a need thereof. Such method is for preventing, delaying and/or treating a disease or a condition associated with an aggressive, inappropriate, and/or disproportionate inflammatory response that can lead to tissue damage. Preferred conditions or diseases are such as allergy, autoimmune disease, or inflammatory disorders of the intestine as already identified herein.

General definitions Sequence identity

"Sequence identity" is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. The whole SEQ ID NO may be used or part thereof. In a preferred embodiment, the whole SEQ ID NO as identified herein is used. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).

Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al, Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. MoI. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. MoI. Biol. 215:403-410 (1990). The well-known Smith Waterman algorithm may also be used to determine identity.

Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. MoI. Biol. 48:443-453 (1970); Comparison matrix: BLOSUM62 from S Henikoff and JG Henikoff, Amino Acid Substitution Matrices from Protein Blocks Proc. Natl. Acad. Sci. USA. 89: 10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program useful with these parameters is publicly available as the "Ogap" program from Genetics Computer Group, located in Madison, WI. The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps). Preferred parameters for nucleic acid comparison include the following:

Algorithm: Needleman and Wunsch, J. MoI. Biol. 48:443-453 (1970); Comparison matrix: matches=+10, mismatch=O; Gap Penalty: 50; Gap Length Penalty: 3. Available as the Gap program from Genetics Computer Group, located in Madison, Wis. Given above are the default parameters for nucleic acid comparisons.

Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gin or his; Asp to glu; Cys to ser or ala; GIn to asn; GIu to asp; GIy to pro; His to asn or gin; He to leu or val; Leu to ile or val; Lys to arg; gin or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb "to consist" may be replaced by "to consist essentially of meaning that a molecule or a viral-based vector or a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. The word "about" or "approximately" when used in association with a numerical value (about 10) preferably means that the value may be the given value of 10 more or less 1% of the value. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

Unless stated otherwise, the practice of the invention will employ standard conventional methods of molecular biology, virology, microbiology or biochemistry. Such techniques are described in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual (2^nd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; in Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA; and in Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK); Oligonucleotide Synthesis (N. Gait editor); Nucleic Acid Hybridization (Hames and Higgins, eds.).

Description of the figures Figure 1.

QPCR and micro-array data for six genes. Validation of microarray results by QPCR using total RNA from biopsies from all eight persons.

Figure 2.

Representative images of laser-capture microdissection (LCM) treatments of cryo- coupes from biopsies, displaying the "harvesting" of intestinal cells captured from stained sections, a) cryo-coupe of tissue before LCM treatment, b) area where epithelial cells will be transferred from the tissue to capture "caps" by the laser treatment; the dark line in the top is the edge of the cap. c) captured epithelial cells as they appear in the cap. d) remaining tissue from image a) after laser capture.

Figure 3. Venn diagrams of bacterial treatments versus placebo (a) and versus one another (b). The numbers indicate significantly regulated genes that are unique for a specific comparison or shared between comparisons.

Figure 4.

Cellular pathways induced after 6 hrs exposure of healthy adult humans to different stages of Lactobacillus plantarum. The horizontal line indicates significance threshold (corresponding to P<0.05); pathways exceeding the threshold are statistically significantly represented according to Fisher's Exact test (calculated in Ingenuity Pathway Analysis) .

Figure 5.

NF-κB subunits and NF-κB signalling or transcription factor activity blockers that are upregulated in healthy adult humans in response to commensal Lactobacillus plantarum bacteria. 5 a, NF-κB subunits and inhibitors upregulated in response to stationary bacteria; 5b, subunits and inhibitors upregulated in response to dead bacteria. Note the activation of NF-κB through association of TNF-α with a TNFR. 5c, inhibitors and TNFR upregulated in response to midlog bacteria.

Figure 6.

Proposed model of cellular proteins and processes corresponding with the presented transcriptional profiles that establish immune tolerance in healthy adults in response to L. plantarum. The genes encoding interleukins are upregulated during the interaction with dead bacteria.

Example Results

Here, we present epithelial transcriptional profiles of the duodenal part of the small intestine of healthy human adults that are altered in response to six hours of interaction with Lactobacillus plantarum. L. plantarum is a bacterial species that does not present any danger to human (MSDS, Public Health Agency Canada). None of the volunteers experienced any discomfort or illness during or after the six- hour oral intake period. Each volunteer consumed about a total of 2xlO^π L. plantarum bacteria. Global array data analysis shows that the same bacterial species in different growth stages induces differential responses in human epithelia

In order to extract as much as possible biological information from the array studies, bacterial treatments were compared against placebo and the three different bacterial treatments were also compared directly against each other.

Complete array gene datasets were used as input in ErmineJ (Lee et al., 2005) for global comparisons; genes that were differentially expressed according to a paired litest using an improved Bayesian algoritm (P <0.05; see Methods) were used to reconstruct cellular pathways using Ingenuity Pathway Analysis. Through this combination of global and specific analysis, it was possible to i) compare global transcriptional profiles between sets of expressed genes per bacterial challenge, and ii) to compare altered expression of clusters of genes grouped together in cellular pathways. According to the ErmineJ analysis output, compared to placebo treatment, different bacterial growth stages induce different altered transcriptional profiles in human epithelia in terms of cellular localisation, biological process and molecular function (Table 1). Whereas dead bacteria (grown to stationary phase and killed by heating to 80 ⁰C) induce mainly responses correlating with innate and adaptive host immune responses, living bacteria induce processes that stimulate not only immune responses but also more basal cellular activity and metabolism. Gene set enrichment analysis (GSEA; Subramanian et al., 2005) showed that, compared to placebo treatment, epithelia responded upon challenge by dead bacteria with expression profiles most similar to those obtained under conditions of maturation of monocyte-derived dendritic cells, tissues responding to interferons and TNF-α and IL-I (not shown). Notably, there is a pronounced difference in responses to L. plantarum bacteria harvested at different phases of bacterial growth, i.e., midlogarithmic ("midlog") and stationary phase of growth ("stationary bacteria"). Responses to stationary bacteria are mainly associated with stimulation of cellular physiology in response to extracellular stimuli including bacteria (GO annotation) and are most similar to GSEA gene sets involving NF-κB-dependent responses to TNF-α, responses to oxidative stress, hypoxia and ultraviolet light, B cell activation and resistance to drugs (not shown). In contrast, responses to midlog bacteria are mainly associated with rRNA processing and rRNA and tRNA metabolism, cytoplasm organisation and biogenesis including ribosome biogenesis and assembly. The most enriched gene sets (GSEA) included (not shown) genes modulated during autophagy, hypoxia, genes regulating the cell cycle and differentiation of T cells, and during growth and differentiation in response to MYC (not shown). Notwithstanding these differences, biological processes induced by living bacteria are more similar than those induced by the dead bacteria that mainly induce immune response-associated cellular pathways. Interestingly, the molecular functions induced by dead and stationary bacteria are more similar, suggesting that, although the cellular pathways induced by these two bacterial preparations are similar, the outcome of these pathways is different in terms of biological processes. To further explore the specificity of human epithelial responses to bacterial growth stage, epithelial responses to these different stages were also directly compared against each other, excluding the placebo intervention from the comparative analysis.

The differential induction of immune response-related genes by bacterial preparations that differ in terms of stage of growth and live or dead status is more pronounced in these global inter-comparisons (Table 2). In the inter-comparison of dead and stationary bacteria, Ig-mediated immune responses and MHC activity together with leukocyte and T cell activation and regulation of immune and defence responses show highest differential regulation. GSEA showed that the most similar gene sets involved processes such as inflammation, responses to dsRNA, interferon, IL-6 and TNF-α, maturation of monocyte-derived dendritic cells and of T cells (not shown). The largest difference between mid-log and dead bacteria is the differential regulation of cytoplasm biosynthesis and nucleic acid metabolism associated with cellular proliferation that is induced in epithelia as a result of the interaction with mid-log bacteria (Table 1). Enriched gene sets include those involved in cell cycle control, growth and proliferation and T cell differentiation (not shown). The most important difference between the response to preparations containing mid-log and stationary bacteria is the induction of immune response-related genes which is a feature of epithelial responses to the stationary bacteria rather than to mid-log bacteria (Table 1). Also in this comparison, GSEA showed that the most similar gene sets were involved in cell growth, proliferation and and differentiation including plasma (B) cell differentiation, inflammation, autophagy, hypoxia and proteasome activity (not shown). These altered transcriptional profiles show that modulation of immune responses, together with the stimulation of cellular proliferation, metabolism and biogenesis are important features of epithelia of healthy humans upon challenge by L. plantarum. It also shows that epithelia are able to distinguish between different preparations of the same bacterial strain.

Laser-capture microdissection and QPCR validate array data and show that relative expression of genes specific for epithelia or lamina propria is differential Quality control of the array hybridisations and primary data analysis was performed using the basal Affymetrix QC and additional Bioconductor packages according to the most recent criteria (see "Methods") in R (R Development Core Team, 2007). This stringent QC ensured that the array hybridisations were of the highest possible quality, and that the statistical tests were able to reliably retrieve differentially expressed genes.

In order to validate the gene expression measured by the micro-arrays, quantitative PCR (QPCR) was performed for each person, using total RNA from biopsies or RNA from epithelial cell pools enriched by laser capture microdissection (LCM). Using freshly isolated biopsy total RNA, the QPCR data validate for each person the expression levels as measured by the micro-arrays for multiple upregulated genes, selected on their relatively high fold-change, low P-value and biological relevance (Figure 1). Compared to upregulated genes, downregulated genes tended to be less consistently expressed compared between persons, did show higher P-values in t-tests and were less well validated by QPCR (Figure 1, SLPI).

The specificity of gene expression in epithelia and the underlying lamina propria, the region harbouring immune cells, was evaluated using cell pools strongly enriched for epithelial cells that were obtained by laser capture microdissection (LCM; Espina et al., 2006). Based on microscopic inspection, the LCM-enriched cell pools consisted for ca. 90% of epithelial cells (Figure 2). Expression of multiple genes selected on their predicted differential expression in epithelia or lamina propria was studied by QPCR. 30 high-quality RNA samples were obtained from epithelial cell pools and compared with the corresponding RNA samples from biopsies of the eight persons. The expression levels of the small intestinal epithelial-specific aquaporin-10 (AQPlO) gene were two-fold higher in the LCM enriched epithelial cell-derived RNA samples compared to the RNA isolated from total biopsies (not shown). Likewise, the expression levels of EGLN3, CXCL2 and CD55 were twofold higher, and of SLCl 1A2 1.5-fold higher in the epithelial cells enriched preparation in comparison to the total biopsy tissue. Conversely, we expected that immune response-related genes, expecially expressed by cells in the lamina propria, would show relatively lower expression in epithelial cell pools. Indeed, expression of CCL20 and PSMB8 was lower in the LCM- enriched epithelial cell pools compared to biopsy total RNA (not shown).

The possibility to perform LCM allowed us to evaluate whether gene expression in a specific part of the biopsy tissue could be correlated to a specific biological function of that tissue. Microarray and QPCR results using RNA from biopsies or LCM-enriched epithelial cell pools showed that expression of the genes SLCl 1A2, EGLN3, CXCL2 and CCL20 was upregulated in epithelia after the different bacterial challenges compared to placebo treatment. Since especially the gene products of CXCL2 and CCL20 have immune response-related properties, it was expected that, after induction by bacteria, higher amounts of CXCL2 (expressed by monocytes, neutrophils and corneal epithelial cells) and CCL20 (expressed by DCs, macrophages, monocytes and IECs) RNA transcripts were present in RNA samples from total biopsies that include the immune cell-rich lamina propria, compared to LCM-enriched epithelial cell pools. Indeed, a proportionally stronger increased transcription of the cytokine-encoding genes CXCL2 and CCL20 was found in the RNA samples from the complete biopsies. In contrast, transcription of the genes SLCl 1A2 and EGLN3 was proportionally stronger increased in the epithelial cell pools (not shown). As a further reference comparison, relative expression of the mildly responsive CD55 gene (Figure 1) was studied. Expression of CD55 was only significantly altered (slightly down- regulated) after oral intake of stationary bacteria and therefore, appeared to be less responsive to bacterial challenge. As might be expected, no altered relative expression was found for this gene in the enriched epithelial cell pool or total biopsy tissue after bacterial challenge (not shown).

Specific immune responses and cellular pathways that are altered in epithelia responding to L. plantarum bacteria The global Ermine J analysis does not include statistical testing for significant differences in gene expression between groups. Therefore, paired t-tests using a specialised Bayesian algorithm (see Methods) were performed to identify genes that were significantly (P<0.05) differentially expressed in epithelia responding to different bacterial growth stages. Between 400 (stationary-placebo) and 800 (midlog-placebo) genes were differentially regulated in response to bacterial challenge, with a trend towards down-regulation (between 55% and 65%, Table 3, Figure 3). The largest fold- changes were between -6.5 and 10.5, in the comparison dead-placebo (Table 3). In order to compare significantly different transcriptional profiles corresponding to specific bacterial challenges in a biological meaningful way, co -transcribed genes were grouped together into known cellular pathways and processes that describe cellular states. The most common pathways induced by bacterial challenge were associated with immune responses, activation of signalling pathways, cell death modulation and processes involved in lipid and fatty acid metabolism (Table 5, Figure 4). As observed in the global Ermine J analysis, epithelial responses to dead and stationary-phase bacteria were more similar to each other and clearly set apart from response to mid log bacteria. Whereas stationary and especially dead bacteria induced multiple processes that are part of the human immune response, mid log bacteria primarily induced processes associated with an increase in cellular metabolism and biogenesis. Pivotal in this comparison was the differential induction of NF-κB subunits by the three bacterial stages. NF-κB signaling upon bacterial stimulation of receptors such as Toll-like receptors (TLRs) and NODI/2 is among the major cellular responses to bacterial challenge (Chen and Greene, 2004; Hayden et al, 2006; West et al, 2006). NF-κB consists of multiple subunits, and the composition of this transcription factor- complex determines its functionality (Hayden and Gosh, 2004; Wietek and O'Neill, 2007). Specifically the p50/p65 NF-κB heterodimer transcription factor complex is correlated with driving the transcription of genes that promote inflammation and cell death in presence of TNF-α and in the absence of inhibitory factors such as BCL-3 or IKB (Hayden and Gosh, 2004; Wietek and O'Neill, 2007). The p65 subunit (ReIA) was not differentially expressed in response to interaction of the mucosa with L. plantarum (Table 4) but was expressed at relatively low levels in all persons. Preparations of dead bacteria induced increased expression of nearly all NF-κB subunits, including elevated expression of the NF-κB signaling inducer TNF-α, but also the inhibitory BCL-3, A20 and IKB (Table 4, Figure 5a). Preparations of stationary bacteria induced expression of p50/p52 NF-κB subunits and the same three inhibitors as dead bacteria but did not induce expression of TNF-α (Table 4, Figure 5b). Strikingly, mid-log bacteria induced only expression of the two NF-κB-inhibitory subunits IKB and BCL-3 (Table 4, Figure 5c). Accordingly, expression of NF-κB controlled genes such as cytokine CXCL2 (Carmody et al., 2007) was not significantly increased across eight persons after challenge with mid-log bacteria, whereas it was among the genes with highest increased expression levels in all persons after challenge with stationary or dead bacteria (Figure 1). Rather than inducing immune responses downstream of NF-κB signalling, responses to mid-log bacteria comprised processes involved in cellular proliferation, such as positive control of cell cycle regulation, nucleic and amino acid metabolism, stimulation of connective tissue development and overall cellular development (not shown and Figure 4;:).

Genes essential in mediating immune tolerance are regulated during the interaction of human epithelia and L. plantarum

Several genes are known to be involved in achieving immune tolerance and display differential expression in persons suffering from IBD including Crohn's disease. Genes such as A20 (TNF AIP3), TREM1-2 and TREML1-2, BCL3, SOCS3 and SLPI are necessary to avoid overt immune responses. The appropriate level of expression of these genes appears not to be reached in persons suffering from Crohn's disease (Haller and Jobin, 2004; Clavel and Haller, 2007; Xavier and Podolsky, 2007). Other genes, such as the genes encoding APRIL (TNFS 13), BAFF (TNFS 13B) and TSLP, stimulate or amplify inflammatory responses and proliferation and survival of tissue-damaging immune cells. The genes encoding A20, BCL3, SOCS3, TREMLl, SLPI, and APRIL were differentially regulated as part of the response of the intestinal mucosa of healthy humans to L. plantarum (not shown). The genes with immunoprotective functions A20, BCL3, and SOCS3 were all up-regulated together with the NF-κB-dependent pro- inflammatory pathways that they antagonise (Table 4 and not shown ). The gene encoding APRIL, an immune response-stimulatory factor that is produced by DCs (MacLennan and Vinuesa, 2002) was up-regulated in response to stationary bacteria; BAFF was not expressed (not shown). The co-stimulatory immunoreceptor TREMLl was down-regulated in response to dead and mid-log bacteria, whereas SLPI was down-regulated upon exposure to stationary and dead bacteria. SLPI and TSLP together regulate class-switching of IgA leading to production of non-specific, broadly reactive IgA and IgG antibodies, a process that is also dependent upon the enzyme AID (Xu et al., 2007). No expression of TSLP and AID was found in response to bacterial challenge (not shown). Finally, immunoglobulins (Igs) that are produced to protect mucosal surfaces from microorganisms show a differential expression pattern in response to the three different preparations of L. plantarum. IgD (heavy-chain) was down-regulated during the interaction with dead bacteria, and not regulated in response to stationary or mid-log bacteria. IgJ was up-regulated by mid-log bacteria exclusively. The main subclass Ig of human jejunum, IgAl (Blum and Schiffrin, 2003) showed a non-significant trend towards increased expression in response to dead (P=O.16), stationary (P=O.37) and mid-log (P=O.07) bacteria.

Discussion

Here, we present transcriptional profiles of human intestinal epithelia that are altered in response to non-pathogenic lactic acid bacteria. Instead of using a mouse model or human cell line, these altered profiles were obtained in vivo during a placebo-controlled double-blind cross-over study involving healthy human volunteers in response to three different preparations of the non-pathogenic food-grade bacterium Lactobacillus plantarum. One preparation consisted of bacteria harvested during mid- logarithmic (exponential) growth, here indicated with "mid-log bacteria"; one preparation consisted of bacteria harvested during stationary-phase growth, here indicated with "stationary bacteria". "Dead" bacteria, finally, are stationary bacteria that were killed by a heat treatment. This study shows, for the first time, that oral administration of nonpathogenic bacteria can in vivo modulate transcriptional profiles of the proximal small intestinal mucosa in healthy humans, regulating innate and acquired immune responses, pro-inflammatory as well as regulatory cytokines and pathways known to be involved in inflammation and its control. These profiles demonstrate that, in response to L. plantarum, genes with known functions in dampening innate immune responses and prevent overt adaptive immune responses are up-regulated, whereas genes that are necessary for establishing and amplifying inflammatory immune responses and proliferation of cytotoxic immune cells are not expressed. The transcriptional profiles presented here may therefore be seen as an expression "blueprint" for establishing immune tolerance in healthy humans.

Laser capture microdissection contributed to measuring gene expression in cells pools enriched for IECs The biopsies that were used in this study contained a mixture of epithelial cells and immune cells from the lamina propria; presence of muscle tissue cells can not be excluded. It was expected that expression of genes that are mainly expressed in lamina propria but less in the better represented epithelial cells would result in an overall lower expression being measured by the array platform. To evaluate the relative expression of genes typically expressed in the epithelial cells or lamina propria, laser capture microdissection (LCM) was performed to obtain cell pools enriched in epithelial cells. RNA was extracted from this enriched collection and QPCR-amplified. Indeed, in these enriched pools, higher fold-changes were found for genes that are specifically expressed in epithelial cells (not shown). This corroborates earlier reports showing that LCM-QPCR contributes to finding expression of genes that are only expressed in specific cell types (e.g. Hooper et al, 2003). Conversely, RNA encoding cytokines typically produced by immune cells was extrapolated to be present at lower levels in LCM-enriched epithial cell pools, relative to the total RNA samples that also contained the RNA from the immune cell-rich lamina propria in addition to RNA from the epithelial cells (not shown).

Differential immune responses induced by different preparations of L. plantarum are presumably caused by cell wall and membrane composition The cell wall of L. plantarum is composed of a mixture of peptidoglycan (44%) and a moiety of teichoid acids coupled to a polysaccharide fraction containing rhamnose (28%) and a second polysaccharide fraction without rhamnose (20%), both containing D-alanine; and a protein (8%) fraction that is associated with the wall teichoic acids (Ikawa, 1961). The D-alanine is important since it stimulates pro-inflammatory immune responses of peripheral blood mononuclear cells and monocytes cultured in vitro (Grangette et al., 2005). The L. plantarum wall and membrane fractions are both of serological relevance. Antigenicity of the wall ribitol teichoic acid is dependent on its association with the protein fraction; the membrane glycerol teichoic acids can be targeted by specific antibodies against both the glucose and glycerol components (Knox and Wicken, 1972). Intact L. plantarum bacteria induce, upon intravenous injection in rabbits, antisera against both the wall and membrane teichoic acids. Antisera against the L. plantarum cell wall fraction contain roughly equal amounts of IgG and IgM antibodies; antisera against the membrane fraction contain more variable amounts (between 20 and 50%) of IgM antibodies (Knox and Wicken, 1972). The wall teichoic acids of L. plantarum remain antigenic upon cell disintegration (Knox and Wicken, 1972). In this context, note that a heat treatment (80 ⁰C, 10 min) was performed in order to obtain preparations with dead bacteria. This heat treatment leads to loss 50 to 90% of carbohydrates from Lactobacillus cell wall preparations (Campbell et al., 1978). Furthermore, heat treatment of preparations of Lactobacillus casei in 0.85% NaCl leads to hydrolysis of the phospho-diester bond between polysaccharide and peptidoglycan, resulting in the release of immuno -reactive 7V-acetylhexosamine (Campbell et al., 1978). It is therefore very likely that the heat treatment of stationary L. plantarum in standard growth medium at 80 ⁰C, performed in order to get the preparation of dead bacteria, has led to similar modification of peptidoglycan and teichoid acids and thus, altered immunogenicity. The heat treatment may have led to the release of immunogenic L. plantarum wall components, providing easier accessible immunogenic components to the human immune system, for instance D-alanine- containing teichoid acids fragments (Wicken et al. 1982a, b; Grangette et al., 2005). Presence of relatively more, easier accessible immunogenic wall fragments presumably explains the stronger induction of immune responses by heat-killed stationary L. plantarum as compared to the living stationary bacteria.

Differential induction of immune responses by living and dead L. plantarum has been found earlier. Bloksma et al. (1979) described that whereas preparations of viable L. plantarum induced mild inflammatory responses, dead bacteria induced mainly antibody formation of immune cells when provided as an adjuvant together with sheep red blood cells in mice. In addition, subcutaneous injection of living bacteria induced infiltration of mainly mononuclear immune cells (such as monocytes and macrophages) (Bloksma et al., 1979, 1981), whereas injection of dead L. plantarum stimulated infiltration of equal numbers of polymorphonuclear and mononuclear cells (Bloksma et al., 1979b). Especially polymorphonuclear cells (that include the neutrophils) are associated with inflammatory responses. The findings of Bloksma and colleagues, although obtained in mice, in different tissue and under different experimental conditions, are in agreement with the transcriptional profiles we describe here. We have shown that only administration of dead L. plantarum leads to increased expression of TNF-α, hallmark of an inflammatory response. In addition, we have shown that only administration of preparations of dead L. plantarum induces genes that are involved in antibody presentation by B cells and maturation of immuno-competent T cells and acute inflammatory responses (Table 1). This induction of genes involved in activation of T cells and antigen processing and presentation (Table 1) is also in agreement with Bloksma et al. (1979) who reported that only administration of preparations of dead L. plantarum induce antibody-presenting cells in mice.

NF-κB subunits and controlling factors are differentially induced by different preparations of L. plantarum

One striking feature was the differential regulation of NF-κB subunits in response to different stages of L. plantarum. NF-kB is typically induced by TLRs and NOD 1/2 (Hayden and Ghosh, 2004; West et al., 2006). L. plantarum wall fragments stimulate intracellular NOD2 (Hagasaki et al., 2006) and TLR2 and TRL4 (et al., 2002, 2004; Marco et al., 2006). In THP-I cell lines, lipoteichoic acids from L. plantarum can modulate the expression of IL-10 and IL-23 as well as induce an increased expression and secretion of TNF-α (Kim et al., 2007). TNF-α, IL-12 and interferon-γ are also stimulated in peripheral blood mononuclear cells (Grangette et al., 2005).

In this study, during the interaction of dead and stationary bacteria with epithelia, several NF-κB subunits were significantly up-regulated (Table 4). NF-κB signalling and nuclear translocation of the heterodimerised NF-κB subunits p50/p65 is a major cause of elevated transcription of genes involved in inflammatory responses. However, the p65 subunit (ReIA) was not regulated and expressed at relatively lower levels (Table 4: NF-κB subunits). Instead, the genes encoding p50/p52 and RelB/p52 complexes (the latter only during interaction with dead bacteria) were transcribed at significantly higher levels. The RelB/p52 dimer (which originates from a processed RelB/plOO dimer) drives transcription of genes involved in lymphoid organogenesis (Wietek and O'Neill, 2007) and may be involved here in "Immune and lymphatic system development and function" which is no. 5 in the list of most regulated cellular functions that are induced during the response of epithelia to dead L. plantarum (not shown). Of the receptors leading to downstream NF-κB signaling, only CD 14 was up- regulated by dead bacteria.

Modulation of NF-κB signalling is important for maintaining exaggerated or inappropriate inflammatory responses. Indeed, genes modulating NF-KB signalling and restricting or dampening pro-inflammatory innate immune responses were up-regulated together with NF-κB subunits: the genes encoding A20 (TNFAIP3), IKB and the sequence- and functionally-related BCL3 (Table 4). IκBα expression was at least 10- fold higher than IκBβ expression (not shown).

Other genes encode proteins that indirectly regulate immune responses, by amplifying pro-inflammatory signals or mediating the cellular environment, for instance by stimulating calcium signaling. TREMLl contains an immunoreceptor tyrosine inhibitory motif (ITIM) and can recruit phosphatases SHP 1-2. The protein is mainly present in α-granules of megakaryocytes and on megakaryocyte-derived platelet surfaces (Barrow et al., 2004; Washington et al., 2004). TREMLl stimulates calcium signalling via FcεRI, the high-affinity receptor for the Fc region of IgE. Indeed, FcεRI signalling was significantly overrepresented in the list of all cellular responses induced during the interaction with dead bacteria and, to a lesser (not significantly) extent, during the interaction with stationary and mid-log bacteria (not shown). TREMLl may dampen the responsiveness to LPS and DC maturation and is therefore an inhibitory receptor that, via its ITIM motif, co-regulates signalling of the pro-inflammatory TREM family receptors (Washington et al., 2002, 2004).

Other factors interfering with NF-κB signalling downstream from TLRs were also up-regulated. SOCS3 suppresses TLR- induced pro-inflammatory signals by interfering with downstream signalling by abolishing the interaction between TRAF6 and TAKl (Yoshimura et al., 2007). Additionally, expression of SOCS3 by CD4+ T- cells and DCs inhibits inappropriate STAT3 activation and regulates differentiation of T_H2 cells and regulatory T cells (Yoshimura et al., 2007). SOCS3 expression correlates with IL-2 expression in response to dead bacteria and may be important for proper balance between immunocompetent and naive or resting (CD4+) T and B cells and DCs.

No cytotoxic adaptive immune responses are induced upon exposure to L. plantarum

The expression of factors that amplify or co-stimulate inflammatory responses and maturation or survival of potentially cytotoxic immune cells was hardly regulated during the interaction with commensal L. plantarum. The epithelial cytokine TSLP stimulates DCs to induce class switching and ultimately, to secrete polyactive IgG and IgA antibodies by activated B cells (Xu et al., 2007). TSLP was not expressed in response to commensal L. plantarum bacteria and only expressed at very low levels in all persons (not shown). Inappropriate expression of TSLP leads to prolonged survival of CD4+ T cells (that may develop in response to IL-2 secretion by DCs) and may eventually develop into of CD4+ T cell-mediated allergic inflammations (Ziegler and Liu, 2006). AID, APRIL and BAFF are factors that, together with TSLP, modulate function and activity of DCs and T and B cells such that polyreactive antibodies are produced (Xu et al., 2007). Of these factors, only APRIL was stronger transcribed in response to stationary bacteria, whereas the other factors did not show increased expression (not shown). A comprehensive overview of expression profiles of these immune cell-modulating factors shows that these profiles are not consistent with a systematic promotion of pro-inflammatory immune responses including production of polyreactive Ig antibodies (Figure 6). Rather, the expression profiles of epithelia in response to commensal L. plantarum are consistent with balanced responses as can be found during an elevated state of physiological inflammation (Sansonetti and di Santo, 2007).

No significant increased expression of IgA was observed in this study. A specific fraction of total IgA is produced to protect mucosal surfaces against resident commensal microbiota by a T-cell-independent, B cell-dependent process (MacPherson et al., 2000). The fact that IgA expression is not significantly increased after oral intake of 10¹⁰ bacteria may reflect the tremendous buffering capacity of colonised duodenum in healthy humans.

Midlog L. plantarum may stimulate cell growth and proliferation

The epithelial response to mid-log L. plantarum bacteria is unique in the up-regulation of MYC (c-Myc) and cyclin Dl, both potent positive regulators of cell proliferation. These findings, together with up-regulated cellular functions and pathways including Wnt/β-catenin signalling and nucleic acid and amino acids metabolism (not shown), suggest that mid- log bacteria stimulate proliferation in the epithelial layers. This stimulation may be due to production of lactate, which is highest in the exponentionally growing mid-log bacteria. The transcriptional profiles that we obtained show that one class of genes that is induced after administration of stationary bacteria is positive regulation of organismal physiology, and that administration of mid-log bacteria leads to induction of genes involved in cytoplasm organisation and ribosome organisation and biogenesis together with tRNA and rRNA metabolism. These processes may have been controlled through the up-regulated MYC and cyclin D l . Interestingly, L. plantarum has previously been shown to be able to stimulate cell growth and proliferation of the mouse spleen and liver (Bloksma et al., 1979b) suggesting that this bacterial species is able to stimulate cellular proliferation of different animal tissues. It is less likely that the differential immune responses are growth-dependent since the immunogenicity of L. plantarum teichoic acids is not influenced by the bacterial growth rate (Knox et al., 1979).

Concluding remarks

This unique in vivo study employed a double-blind placebo-controlled randomised cross-over design to investigate human responses to a well-studied food-grade bacterium that has been the subject of diverse biochemical and immunological reports since the early 1960's. One major finding was the difference in epithelial responses to different preparations of L. plantarum, especially the much weaker immune response to mid-log bacteria compared to stationary and dead bacteria.

Materials and Methods

Preparation of bacterial interventions

For the preparation of the bacterial preparations used as live- and dead- stationary phase cells, a 500 ml full-grown cultures of Lactobacillus plantarum WCFSl (overnight in MRS (de Man, Rogosa and Sharpe) from Merck, Cat. No. 1.10660., at 37 ⁰C) was used to inoculate 16 L of fresh and pre-warmed MRS medium and cultured overnight (37 ⁰C, stationary phase cultures). Cells were harvested by centrifugation (5000rpm, 20 min), cells were washed once with MiIIiQ water, and re-suspended in 600ml MiIIiQ. This suspension was divided in two equal portions (350ml each), and one of these portions was heat-treated (10 min 85 ⁰C) to prepare the so-called "dead" bacterial preparation. To both portions (live and dead-stationary phase cells) maltodextrin and glucose were added to a final concentration of 20 % (W/V) and 2% (W/V), respectively. Bacterial suspensions were divided over 10 tubes with 3 ml per tube, and 130 tubes with 2 ml per tube and frozen at -40 ⁰C and subsequently freeze- dried. For the preparation of the mid-logarithmic growth phase L. plantarum cultures, a 250 ml full-grown cultures of Lactobacillus plantarum WCFSl (overnight in MRS [Merck] medium, at 37 ⁰C) was used to inoculate 16 L of fresh and pre-warmed MRS medium and cultured (37 ⁰C) until an optical density at 600 nm of 1.0 was reached (actual OD600 was 1 .017; mid- logarithmic growth phase). Cells were harvested by centrifugation (5000rpm, 20 min), washed once with MiIIiQ water, and re-suspended in 300 ml MiIIiQ. Maltodextrin and glucose were added to a final concentration of 20 % (W/V) and 2% (W/V), respectively. Bacterial suspensions were divided over 10 tubes with 3 ml per tube, and 130 tubes with 2 ml per tube and frozen at -40 ⁰C and subsequently freeze-dried.

Placebo intervention materials were prepared by dissolving maltodextrin 20 % (W/V) and glucose 2% (W/V), in 300 ml of MiIIiQ. The solution was divided over 10 tubes with 3 ml, and 130 tubes with 2 ml per tube and subsequently freeze dried. Freeze-dried bacterial preparations were stored at 4 ⁰C in tightly closed tubes until use. Bacterial viable counts were determined for each of the bacterial preparations both at the beginning of the intervention trial as well as at the end of the entire trial (after 4 interventions were completed in all subjects) and appeared to be virtually unaffected by the storage period. Viable counts were determined as colony forming units on MRS- agar plates. The freeze dried stationary-phase bacterial preparation contained approximately 2 x 10¹⁰ cfus per ml, while no cfus could be detected in the heat-treated variant of the same bacterial suspension, nor in the placebo preparations. The mid- logarithmic bacterial preparation contained approximately 1.7 xlO¹⁰ cfus per ml. Viable counts are expressed as cfu per ml of original suspension, prior to freeze-drying, but were determined in the freeze-dried preparations. Freshly prepared suspensions or placebo drinks were administered 1 x 150 ml (from 3 ml freeze-dried tube) at the start of the intervention, followed by 12 xlOO ml (from 2 ml freeze-dried tube) every 30 minutes, over a period of 6 hours. » biopt.

Volunteers This human intervention study was approved by the University Hospital Maastricht Ethical Committee, and conducted in full accordance with the principles of the 'Declaration of Helsinki' (52nd WMA General Assembly, Edinburgh, Scotland, Oct 2000). All subjects gave their written informed consent prior to their inclusion into the study. Eight healthy non-smoking volunteers (24 ± 4y) without a history of gastrointestinal symptoms and free of any medication, were investigated on four separate occasions in a randomized placebo-controlled crossover study. On a single occasion, volunteers were provided with bacterial preparations that were reconstituted from freeze dried bacteria and resuspended in maltodextrin solution just prior to oral intake, or only containing the maltodextrin solution (the placebo control). During a total period of six hours, volunteers ingested bacterial preparation or placebo control each half hour. Both the volunteers and the researchers providing the beverages did not know whether a subject received a bacterial preparation or a placebo control during the experimental period ("double-blind" study). After this 6-h period, tissue samples were obtained from the horizontal part of the duodenum by standard flexible gastroduodenoscopy, at approximately 15 cm distal to the pylorus. In all tissue samples, gene expression levels were measured using genome-wide microarrays (Affymetrix U133A Plus2 arrays).

RNA isolation and quality control

Total RNA was extracted from biopsies with TRIzol reagent, purified and DNAse treated using the SV Total RNA Isolation System (Promega, Leiden, The Netherlands). RNA integrity was checked on an Agilent 2100 bioanalyzer (Agilent Technologies, Amsterdam, the Netherlands) with 6000 Nano Chips according to the manufacturer's instructions. RNA was judged as suitable for array hybridization only if samples showed intact bands corresponding to the 18S and 28S ribosomal RNA subunits, displayed no chromosomal peaks or RNA degradation products, and had a RIN (RNA integrity number) above 8.0.

Affymetrix GeneChip oligoarray hybridization and scanning

Each of eight volunteers was subjected to four biological treatments: three bacterial interventions and one placebo. Total RNA, extracted from biopsies was labelled using the Ambion Message Amp II biotin enhanced single-round amplification kit (cat. no. 1791). The correspondingly labelled RNA was hybridised to a Gene Chip HuI 33 Plus2 array. Detailed methods for the labelling and subsequent hybridisations to the arrays are described in the eukaryotic section of the Gene Chip Expression Analysis Technical Manual, Revision 3, from Affymetrix (Santa Clara, CA), and are also available upon request.

Analyses and functional interpretation of microarray data Packages from the Bioconductor project (www.bioconductor.org) were used for analysing the scanned Affymetrix arrays (Gentleman et al, 2004). Arrays were normalised using quantile normalisation, and expression estimates were compiled using RMA applying the empirical Bayes approach (Wu et al., 2004). The Bioconductor packages were integrated in the automated on-line MADMAX pipeline

Because it was expected that responses of human epithelia to commensal bacteria would show only a modest increase in gene expression, array hybridisations and data needed to be of very high quality. Arrays were considered of sufficient quality when they showed not more than 10% of specks in fitPLM model images, were not deviating in RNA degradation and density plots, when they were not significantly deviating in NUSE and RLE plots and were within each other's range in boxplots. For a more extensive description of quality criteria, please contact the authors.

Differentially expressed probe sets were identified using linear models, applying moderated t-statistics that implement empirical Bayes regularisation of standard errors (Smyth, 2004). P-values were corrected for multiple testing using a false discovery rate method (Storey et al., 2003); raw P-values < 0.05 were included in further analyses. To be able to include variance of expression data of all four groups (three interventions, one placebo) in paired t-tests, the novel IBMT procedure (Sartor et al., 2007) was also used to see whether inclusion of extra variance would influence the outcome of paired t-tests.

Three complementary methods were applied to relate changes in gene expression to functional changes. One method is based on overrepresentation of Gene Ontology (GO) terms (Lee et al., 2005). Another approach, gene set enrichment analysis (GSEA), takes into account the broader context in which gene products function, namely in physically interacting networks, such as biochemical, metabolic or signal transduction routes (Subramanian et al., 2005). Both applied methods have the advantage that they are unbiased, because no gene selection step is used, and a score is computed based on all genes in a particular GO term or gene set. In addition, biological cellular functions activated in response to bacterial interventions were identified using Ingenuity Pathways Analysis (IPA) (Ingenuity Systems, Redwood City, CA). Input gene lists included those genes of which the expression had changed with raw P-values < 0.05. In order to identify major activated pathways, a fold-change cut-off value of 2 was used. In order to reconstruct cellular pathways including those genes that showed significantly altered expression, a fold-change cut-off of 1.1 was used. Small (10-40%) changes in gene expression in human tissue induced by mild stimuli have been published earlier (Mootha et al, 2003; Patti et al, 2003) and may be a characteristic of mean expression changes in human tissue, typically consisting of multiple different cell types with possibly differing gene expression programs.

Cellular pathway reconstruction and biological interpretation

Lists of up- and down-regulated genes, their fold-change (FC) and P-values were used as input in the software package Ingenuity Pathways Analysis (IPA)

IPA annotations follow the GO annotation principle, but are based on a proprietary knowledge base of over 1,000,000 protein-protein interactions. The IPA output includes metabolic and signalling pathways with statistical assessment of the significance of their representation being based on Fisher's Exact Test. This test calculates the probability that genes participate in a given pathway relative to their occurrence in all other pathway annotations.

Preparation of tissue sections and LCM

Frozen sections (7 μm thickness) were cut at -20⁰C and five sections from one sample were transferred onto a glass slide (Superfrost plus, Fisher). The glass slide was transferred to a microslide box kept on dry ice and stored at -80⁰C. Immediately before LCM, the glass slide with the frozen sections was taken from the freezer and thawed for 1 min at room temperature. The section was fixed in 70% ethanol for 10 sec at room temperature, following by washing in nuclease-free water by dipping 10 times. The slide was stained with Histogen staining solution (Arcturus) for 10 sec and washed again in nuclease-free water. Then the slide was dehydrated in an ethanol/xylene gradient series: 10 dips 75% ethanol, 2 times 10 dips 95% ethanol, 1 min 95% ethanol, 3 times 1 min 100 % ethanol, 3 times 1 min xylene. The sections were dried for 2 minutes at room temperature. A 7.5 μm laser beam was used (90 mW, 4.7 mSec) for laser capture microdissection using the Pix-Cell II apparatus (Arcturus). HS caps (Arcturus) were used for capture. 400-2000 cells were shot for each sample.

RNA was isolated from the LCM sample caps using the PicoPure RNA isolation kit (Arcturus) following manufacturer's recommendations, including a DNAse step.

For a reverse transcription reaction, 200 ng RNA (between 4 and 40 ng RNA isolated from the cells isolated with LCM) was incubated at 65 ⁰C for 5 minutes with 0.5 μg random hexamers (Invitrogen) and 1 μl 10 mM dNTP mixture. After 2 minutes on ice, the following was added: 5 μl 5x first strand buffer, 2 μl 0.1 M DTT, 200 Units Superscript III reverse transcriptase, 40 U RNaseOUT RNase inhibitor (all Invitrogen) and water to a final volume of 20 μl. The reaction was incubated at 50 ⁰C for 60 min. The reaction was inactivated by heating at 70 ⁰C for 15 min. Thus obtained cDNA samples were stored at -20 ⁰C until further use. Quantitative PCR amplification was performed in 96-well plates on a 7500 Fast

System (Applied Biosystems) using TaqMan primers and probes and 1 μl of 10-fold diluted RT product was used as a template. Assays were performed following the predeveloped TaqMan assay reagents protocol (Applied Biosystems) with the following primers and probes : SLC l 1 A2 (HsOO 1 67207_m l ) , EGLN3 (Hs00222966_ml), CCL20 (Hs00171125_ml), CXCL2 (Hs00601975_ml), CD55 (Hs00167090_ml), SLPI (Hs00268206_ml), PFN4 (Hs00380763_ml), AQPlO (Hs01587666_gl) PSMB8 (Hs00544760_gl)and GAPDH (Hs02758991_gl). The indices between brackets (Hs***_xx) refer to the specific TaqMan assay that was used to quantitatively PCR-amplify a specific gene fragment. TaqMan probe sequences and the corresponding gene fragments are given in Table 7 for each gene . The following conditions were used: 50 ⁰C for 2 min, 95 ⁰C for 10 min, then 40 cycles at 95 ⁰C for 15 sec, and 60 ⁰C for 1 min. In each run, 4 standards were included with appropriate dilutions of the cDNA to make a standard curve and to quantitate the samples. All measurements were done in duplicate. The significance of the difference in the gene expression levels between the groups was calculated by performing a paired, 2-tailed t-test on the log-transformed data using Microsoft Excel. TABLE LEGENDS

Table 1.

Global transcriptional profiles represented based on Gene Ontology (GO) annotation as determined in Ermine J analysis of bacterial challenges vs. placebo. CC: cellular components; MF: molecular functions, BP: biological processes.

Table 2.

Global transcriptional profiles represented based on Gene Ontology (GO) annotation as determined in Ermine J analysis of inter-compared bacterial challenges.

Table 3.

Differentially expressed genes (P<0.05) from three comparisons of bacterial interventions versus placebo.

Table 4.

Numbers of differentially expressed genes, the direction (up-/down-regulation) and the fold-change range in six different comparisons (bacterial treatment-placebo and treatment-treatment comparisons).

Table 5.

NF-κB subunits differentially stimulated by TNFR and TLR ligands (TNF-α) and two antagonists (IKB and A20) after different bacterial challenges. Fold-changes and corresponding P-values are indicated; "-" indicates no significantly altered regulation (iBayes paired t-test, significance threshold P<0.05).

Table 6.

Regulation of genes mediating epithelial immune homeostasis and tolerance during the interaction with L. plantarum. AID (AICDA); APRIL (TNFSF 13); A20 (TNF AIP3); BAFF (TNFSF13B); IκBα (NFKBIA); TACI (TNFRSF13B). The numbers indicate the fold-change with corresponding (P -value). Bold typeface denotes statistical significance. 1 very low basal expression (3-5 on unlogged scale). Table 7.

List of Taqman assay kits and probe sequences used for QPCR assays.

Table 8

Identification of the DNA coding sequences and encoded polypeptides present in the sequence listing.

Table 1.

Table 2.

Table 3.

Table 4.

Table 5.

Table 6.

Table 7

The used TaqMan assay IDs and corresponding probe sequences to QPCR-amplify selected genes

Tabel 8

References

Baldwin DN, Vanchinathan V, Brown PO, Theriot JA. (2002) A gene-expression program reflecting the innate immune response of cultured intestinal epithelial cells to infection by Listeria monocytogenes. Genome Biol. 4, R2.

Balfour Sartor R. (2007) Bacteria in Crohn's disease: mechanisms of inflammation and therapeutic implications. J Clin Gastroenterol. 41 (Suppl 1), S37-43.

Baumgart DC, Carding SR. (2007) Inflammatory bowel disease: cause and immunobiology. Lancet 369, 1627-1640.

Barrow AD, Astoul E, Floto A, Brooke G, Relou IA, Jennings NS, Smith KG, Ouwehand W, Farndale RW, Alexander DR, Trowsdale J. (2004) TREM-like transcript-1, a platelet immunoreceptor tyrosine-based inhibition motif encoding costimulatory immunoreceptor that enhances, rather than inhibits, calcium signaling via SHP-2. J Immunol. 172, 5838-5842.

Bloksma N, Van Dijk H, Korst P, Willers JM. (1979a) Cellular and humoral adjuvant activity of mistletoe extract. Immunobiology 156,309-318.

Bloksma N, de Heer E, van Dijk H, Willers JM. (1979b) Adjuvanticity of lactobacilli. I. Differential effects of viable and killed bacteria. Clin Exp Immunol. 37, 367-375.

Bloksma N, Ettekoven H, Hofhuis FM, van Noorle-Jansen L, De Reuver MJ, Kreeftenberg JG, Willers JM. (1981) Effects of lactobacilli on parameters of nonspecific resistance of mice. Med Microbiol Immunol. 170, 45-53.

Campbell LK, Knox KW, Wicken AJ. (1978) Extractability of cell wall polysaccharide from lactobacilli and streptococci by autoclaving and by dilute acid. Infect Immun. 22, 842-851. Cario E, Podolsky DK. (2000) Differential alteration in intestinal epithelial cell expression of toll-like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease. Infect Immun. 68, 7010-7017.

Cario E. (2005) Bacterial interactions with cells of the intestinal mucosa: Toll-like receptors and NOD2. Gut 54, 1182-1193.

Cario E, Podolsky DK. (2005) Intestinal epithelial TOLLerance versus inTOLLerance of commensals. MoI Immunol. 42, 887-893.

Carmody RJ, Ruan Q, Palmer S, Hilliard B, Chen YH. (2007) Negative regulation of toll-like receptor signaling by NF-κB p50 ubiquitination blockade. Science 317, 675- 678.

Chen, L.F., and Greene, W.C. (2004). Shaping the nuclear action of NF-κB. Nat. Rev. MoI. Cell Biol. 5, 392-401.

Clavel T, Haller D. (2007) Molecular interactions between bacteria, the epithelium, and the mucosal immune system in the intestinal tract: implications for chronic inflammation. Curr Issues Intest Microbiol. 8, 25-43.

Dann SM, Eckmann L (2007) Innate immune responses in the intestinal tract. Curr. Opin. Gastroenterol. 23, 115-120.

Eickhoff M, Thalmann J, Hess S, Martin M, Laue T, Kruppa J, Brandes G, Klos A. (2007) Host cell responses to Chlamydia pneumoniae in gamma interferon-induced persistence overlap those of productive infection and are linked to genes involved in apoptosis, cell cycle, and metabolism. Infect Immun. 75, 2853-2863.

Espina V, Wulfkuhle JD, Calvert VS, VanMeter A, Zhou W, Coukos G, Geho DH, Petricoin EF 3rd, Liotta LA. (2006) Laser-capture microdissection. Nat Protoc. 1, 586- 603. Foster TJ (2005) Immune evasion by Staphylococci. Nature Rev Microbiol 3, 948-958.

Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, et al. (2004) Genome Biol 5, R80.

Grangette C, Nutten S, Palumbo E, Morath S, Hermann C, Dewulf J, Pot B, Hartung T, Hols P, Mercenier A. (2005) Enhanced antiinflammatory capacity of a Lactobacillus plantarum mutant synthesizing modified teichoic acids. Proc Natl Acad Sci U S A. 102, 10321-10326.

Haller D, Jobin C. (2004) Interaction between resident luminal bacteria and the host: can a healthy relationship turn sour? J Pediatr Gastroenterol Nutr. 38, 123-136.

Hasegawa M, Yang K, Hashimoto M, Park JH, Kim YG, Fujimoto Y, Nunez G, Fukase K, Inohara N. (2006) Differential release and distribution of Nodi and Nod2 immuno stimulatory molecules among bacterial species and environments. J Biol Chem. 281, 29054-29063.

Hayden, M.S., and Ghosh, S. (2004). Signaling to NF-κB. Genes Dev. 18, 2195-2224.

Hayden, M.S., West, A. P., and Ghosh, S. (2006). NF-κB and the immune response. Oncogene 25, 6758-6780.

He B, Xu W, Santini PA, Polydorides AD, Chiu A, Estrella J, Shan M, Chadburn A, Villanacci V, Plebani A, Knowles DM, Rescigno M, Cerutti A. (2007) Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL. Immunity 26, 812-826.

Hooper LV, Stappenbeck TS, Hong CV, Gordon JI. (2003) Angiogenins: a new class of microbicidal proteins involved in innate immunity. Nat Immunol. 4, 269-273. Ikawa M. (1961) The partial chemical degradation of the cell walls of Lactobacillus plantarum, Streptococcus faecalis, and Lactobacillus casei. J Biol Chem. 236, 1087- 1092.

Kabelitz D, and Medzhitov R (2007) Innate immunity - cross-talk with adaptive immunity through pattern recognition receptors and cytokines. Curr. Opin. Immunol 19, 1-3.

Karlsson H, Hessle C, Rudin A. (2002) Innate immune responses of human neonatal cells to bacteria from the normal gastrointestinal flora. Infect Immun. 70, 6688-6696.

Karlsson H, Larsson P, Wold AE, Rudin A. (2004) Pattern of cytokine responses to gram-positive and gram-negative commensal bacteria is profoundly changed when monocytes differentiate into dendritic cells. Infect Immun. 72, 2671-2678.

Kim HG, Gim MG, Kim JY, Hwang HJ, Ham MS, Lee JM, Hartung T, Park JW, Han SH, Chung DK (2007) Lipoteichoic acid from Lactobacillus plantarum elicits both the production of interleukin-23pl9 and suppression of pathogen-mediated interleukin-10 in THP-I cells. FEMS Immunol Med Microbiol. 49, 205-214.

Kleerebezem, M. et al. (2003) Complete genome sequence of Lactobacillus plantarum WCFSl. Proc Natl Acad Sci U S A. 100, 1990-1995.

Knox KW, Wicken AJ. (1972) Serological studies on the teichoic acids of Lactobacillus plantarum. Infect Immun. 6,43-49.

Knox KW, Campbell LK, Broady KW, Wicken AJ. (1979) Serological studies on chemostat-grown cultures of Lactobacillus fermentum and Lactobacillus plantarum. Infect Immun. 24, 12-18. Kobayashi SD, Braughton KR, Whitney AR, Voyich JM, Schwan TG, Musser JM, DeLeo FR. (2003) Bacterial pathogens modulate an apoptosis differentiation program in human neutrophils. Proc Natl Acad Sci U S A. 100, 10948-10953

Lee HK, Braynen W, Keshav K, and Pavlidis P. (2005) ErmineJ: tool for functional analysis of gene expression data sets. BMC Bio informatics 6, 269.

MacLennan I, Vinuesa C. (2002) Dendritic cells, BAFF, and APRIL: innate players in adaptive antibody responses. Immunity 17, 235-238.

MacPherson AJ, Gatto D, Sainsbury E, Harriman GR, Hengartner H, Zinkernagel RM. (2000) A primitive T cell-independent mechanism of intestinal mucosal IgA responses to commensal bacteria. Science 288, 2222-2226.

MacPherson AJ, McCoy K (2007) APRIL in the intestine: a good destination for immunoglobulin A₂. Immunity 26, 755-757.

MacPherson AJ, Uhr T (2004) Compartmentalization of the mucosal immune responses to commensal intestinal bacteria. Ann. N. Y. Acad. Sci. 1029, 36-43.

Marco, M. L., Pavan, S., and Kleerebezem, M. (2006) Towards understanding molecular modes of probiotic action. Curr. Opin. Biotechnol. 17, 204-210.

McCaffrey RL, Fawcett P, O'Riordan M, Lee KD, Havell EA, Brown PO, Portnoy DA. (2004) A specific gene expression program triggered by Gram-positive bacteria in the cytosol. Proc Natl Acad Sci U S A. 101, 11386-11391.

McHeyzer- Williams M. (2007) Local sentries for class switching. Nat Immunol. 8,

230-232.

Medzhitov R, and Janeway Jr. CA (2002) Decoding the patterns of self and nonself by the innate immune system. Science 296, 298-300. Mohamadzadeh M, Olson S, Kalina WV, Ruthel G, Demmin GL, Warfield KL, Bavari S, Klaenhammer TR. (2005) Lactobacilli activate human dendritic cells that skew T cells toward T helper 1 polarization. Proc Natl Acad Sci U S A. 102, 2880-2885.

Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, Puigserver P, Carlsson E, Ridderstrale M, Laurila E, Houstis N, Daly MJ, Patterson N, Mesirov JP, Golub TR, Tamayo P, Spiegelman B, Lander ES, Hirschhorn JN, Altshuler D, Groop LC. (2003) PGC-I alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat. Genet. 34, 267-273.

Nau GJ, Richmond JF, Schlesinger A, Jennings EG, Lander ES, Young RA. (2002 Human macrophage activation programs induced by bacterial pathogens. Proc Natl Acad Sci U S A. 99, 1503-1508.

Patti ME, Butte AJ, Crunkhorn S, Cusi K, Berria R, Kashyap S, Miyazaki Y, Kohane I, Costello M, Saccone R, Landaker EJ, Goldfine AB, Mun E, DeFronzo R, Finlayson J, Kahn CR, Mandarino LJ. (2003) Coordinated reduction of genes of oxidative metabolism in humans with insulin resistance and diabetes: Potential role of PGCl and NRFl. Proc Natl Acad Sci U S A. 100, 8466-8471.

R Development Core Team (2007). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-projcct.org.

Rinella ES, Eversley CD, Carroll IM, Andrus JM, Threadgill DW, Threadgill DS. (2006) Human epithelial-specific response to pathogenic Campylobacter jejuni. FEMS Microbiol Lett. 262, 236-243.

Sansonetti, PJ. (2006) The innate signaling of dangers and the dangers of innate signaling. Nature Immunol. 1237-1242.

Sansonetti PJ, Di Santo JP. (2007) Debugging how bacteria manipulate the immune response. Immunity 26, 149-161. Sartor MA, Tomlinson CR, Wesselkamper SC, Sivaganesan S, Leikauf GD, Medvedovic M. (2006) Intensity-based hierarchical Bayes method improves testing for differentially expressed genes in microarray experiments. BMC Bio informatics 19, 7:538.

Schnaith A, Kashkar H, Leggio SA, Addicks K, Kronke M, Krut O. (2007) Staphylococcus aureus subvert autophagy for induction of caspase-independent host cell death. J Biol Chem. 282, 2695-2706

Smyth GK (2004) Stat Appl Genet MoI Biol 3, Article3.

Storey JD, and Tibshirani R. (2003) Statistical significance for genomewide studies. Proc Natl Acad Sci U S A 100: 9440-9445.

Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, and Mesirov JP. (2005) Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 102: 15545-15550.

Washington AV, Quigley L, McVicar DW. (2002) Initial characterization of TREM- like transcript (TLT)-I: a putative inhibitory receptor within the TREM cluster. Blood 100, 3822-3824.

Washington AV, Schubert RL, Quigley L, Disipio T, Feltz R, Cho EH, McVicar DW. (2004) A TREM family member, TLT-I, is found exclusively in the alpha-granules of megakaryocytes and platelets. Blood 104, 1042-1047.

West, A.P., Koblansky, A.A., and Ghosh, S. (2006). Recognition and signaling by ToIl- like receptors. Annu. Rev. Cell Dev. Biol. 22, 409-437. Wicken AJ, Broady KW, Ayres A, Knox KW. (1982a) Production of lipoteichoic acid by lactobacilli and streptococci grown in different environments. Infect Immun. 36, 864-869.

Wicken AJ, Evans JD, Campbell LK, Knox KW. (1982b) Teichoic acids from chemostat-grown cultures of Streptococcus mutans and Lactobacillus plantarum. Infect Immun. 38, 1-7.

Wietek C, O'Neill LA (2007) Diversity and regulation in the NF-κB system. Trends Biochem Sci. 32, 301-309.

Wu Z, Irizarry RA, Gentleman R, Martinez-Murillo F, Spencer F (2004) Journal of the American Statistical Association 99, 909-917.

Xavier RJ, Podolsky DK (2007) Unravelling the pathogenesis of inflammatory bowel disease. Nature 448, 427-434.

Xu W, He B, Chiu A, Chadburn A, Shan M, Buldys M, Ding A, Knowles DM, Santini PA, Cerutti A. (2007) Epithelial cells trigger frontline immunoglobulin class switching through a pathway regulated by the inhibitor SLPI. Nat Immunol. 8, 294-303.

Yan F, Cao H, Cover TL, Whitehead R, Washington MK, Polk DB. (2007) Soluble proteins produced by probiotic bacteria regulate intestinal epithelial cell survival and growth. Gastroenterology 132, 562-575.

Yoshimura A, Naka T, Kubo M. (2007) SOCS proteins, cytokine signalling and immune regulation. Nat Rev Immunol. 7, 454-465.

Young KT, Davis LM, Dirita VJ. (2007) Campylobacter jejuni: molecular biology and pathogenesis. Nat Rev Microbiol. 5, 665-679. Zhang X, Wang H, Claudio E, Brown K, Siebenlist U. (2007) A role for the IkappaB family member Bcl-3 in the control of central immunologic tolerance. Immunity 27,

438-452.

Ziegler SF, Liu YJ. (2006) Thymic stromal lymphopoietin in normal and pathogenic T cell development and function. Nat Immunol. 7, 709-714.

Claims

Claims

1. A composition comprising lactic acid bacteria, wherein a substantial amount thereof is in the mid- log phase.

2. A composition according to claim 1, wherein at least 50% of the total lactic acid bacteria number of cells is in the mid- log phase.

3. A composition according to claim 1 or 2, wherein the lactic acid bacteria is in a dry form.

4. A composition according to any one of claims 1 to 3, wherein the composition is capable of inducing a gene expression profile indicative of immune tolerance when administrated to a subject.

5. A composition according to claim 4, wherein the expression of a TREMl gene is down-regulated and/or the expression of a PARPl gene is up-regulated .

6. A composition according to any one of claims 1 to 5, wherein the composition is further characterised by the substantially non-induction and/or non expression of immune response-related genes when administrated to a subject.

7. A composition according to any one of claims 1 to 6, for use as a medicament.

8. A composition according to claim 7, wherein the medicament is for preventing, delaying and/or treating at least one of a condition or disease associated with an aggressive, inappropriate, and/or disproportionate inflammatory response that may lead to tissue damage.

9. A device for administering the composition as defined in any one of claims 3 to 8, comprising a first compartment for holding a moist component, a second compartment for holding a dry composition as defined in claim 3 and a separator for separating said first and second compartment, such that when said separator is at least partially removed, said moist component and said dry composition are permitted to mix to form a mixture, thereby forming an alive composition as defined in claim 1 or 2.

10. Use of a composition according to any one of claim 1 to 8 or of a device according to claim 9 for the manufacture of a medicament for preventing, delaying and/or treating at least one of a condition or disease associated associated with an aggressive, inappropriate, and/or disproportionate inflammatory response that can lead to tissue damage.