Inflammatory bowel disease (IBD) is an autoimmune disease characterized by a chronic inflammation of the gastrointestinal tract mucosa and is related to an abnormal immune response to commensal bacteria. Our aim of the present work has been to explore the levels of antibodies (IgG and IgA) raised against extracellular proteins produced by LAB and its association with IBD. We analyzed, by Western-blot and ELISA, the presence of serum antibodies (IgA and IgG) developed against extracellular protein fractions produced by different food bacteria from the genera
Inflammatory bowel disease (IBD) is an autoimmune disease characterized by a chronic inflammation of the gastrointestinal tract (GIT) mucosa. Depending on the severity and location of the injuries, two main forms are distinguished, Crohn’s disease (CD) and ulcerative colitis (UC). Both are chronic disorders of unexplained origin, in which persistent ulcerations appear in the small or large bowel mucosa. Interestingly, genetic susceptibility only explains up to 23% of the disease, in the case of CD (16% for UC), with the rest being attributed to environmental factors, such as an exacerbated response of the innate immune system to the commensal microbiota [
Experiments in germ-free animals have shown that microbial colonization is crucial in the instruction, maturation, and regulation of the immune system. For instance, the presence of
Implications of microbiota dysbiosis extend beyond the obvious differences in microbial composition from a functional and metabolic point of view. For instance, decreased ratios between
IBD is associated with antibodies raised against extracellular molecules of GIT microorganisms, such as extracellular mannan from
Our aim with the present work has been to explore the levels of antibodies (IgG and IgA) raised against extracellular proteins produced by LAB and its association with IBD. The main results are discussed next.
Six bacterial strains representing microorganisms used in human nutrition, or as probiotics, were used in this study:
As routine culturing, isolated bacterial colonies were recovered on MRSC agar plates from frozen stocks stored at −80°C (MRSC supplemented with 40% (v/v) glycerol). Single colonies were used for inoculating MRSC tubes, which were kept in the anaerobic chamber ON. Fresh MRSC bottles containing different volumes, depending on the experiment (from 50 mL to 400 mL), were then inoculated from the ON precultures (1% v/v). These cultures were incubated in the same conditions up to early stationary phase of growth (from 12 to 48 h depending on the bacterium). Cultures were then centrifuged (10,000 ×g, 30 min), and the supernatants were filtered (0.45
Extracellular proteins were obtained from bacterial supernatants following the protocol described by Sánchez et al. [
For detection of immunoreactive bands within the different extracellular protein extracts, 40
Detection of immunoreactive bands was performed using a collection of sera and consisted of 50 samples from healthy individuals, 37 samples from individuals with CD, and 15 samples from individuals with UC. Approval for the study was obtained from the Regional Ethics Committee for Clinical Investigation (Comisión Asesora de Bioética del Principado de Asturias; Servicio de Salud del Principado de Asturias) and all determinations were performed with fully informed written consent. This collection is deposited in the “Colección del Registro Nacional de Biobancos”, Ref. C.0001263 (
Demographic and clinical parameters of UC patients.
Clinical parameters |
|
---|---|
Men/women | 11/7 |
Age, mean ± sd | 45.25 ± 16.89 |
Disease duration, mean ± sd | 8.37 ± 8.57 |
Age at onset, mean ± sd | 36.88 ± 16.79 |
Familial history | 5 (31.3) |
Location | |
Extensive | 9 (56.3) |
Left-sided | 2 (12.5) |
Proctitis | 5 (31.3) |
Extraintestinal manifestations | |
Arthritis | 4 (25.0) |
Dermatological affection | 4 (25.0) |
Values are
Demographic and clinical parameters of CD patients.
Clinical parameters |
|
---|---|
Men/women, |
23/14 |
Age, mean ± sd | 42.89 ± 12.89 |
Disease duration, mean ± sd | 10.08 ± 8.56 |
Age at onset, mean ± sd | 32.78 ± 12.41 |
Familial history | 8 (21.6) |
Location | |
Ileum | 11 (29.7) |
Colon | 5 (13.5) |
Ileum + colon | 20 (54.1) |
Upper gastrointestinal | 1 (2.7) |
Extraintestinal manifestations | |
Arthritis | 14 (37.8) |
Dermatological affection | 9 (24.3) |
Perianal disease | 12 (32.4) |
Fistula | 15 (40.5) |
Values are
Around one milligram of extracellular protein fraction from LGG was separated in polyacrilamide gels, as described before, using multiple wells. CWH is the only extracellular protein of LGG separated in the gel range of 70–80 kDa, so this zone was cut out of the gels with the help of the pre-stained molecular mass marker, containing a red-labelled band of 70 kDa. Gel slides were placed into dialysis membranes with a cut-off of 8 kDa and 3 mL of electrophoresis buffer were added. Dialysis membranes were set in the electrophoresis chamber and the system was maintained for one hour at a constant voltage of 170 V. Following this procedure, CWH was electroeluted from the gel slides. CWH protein, already in solution, was concentrated in a vacuum device at room temperature (Concentrator 5301, Eppendorf AG, Germany), and finally dialyzed against PBS for 24 h at 4°C (Tube-O-dialyzer devices, G-biosciences, cut-off 1 kDa). Protein concentration was determined by the bicinchoninic acid test (BCA kit, Thermo Fisher Scientific), using a series of bovine serum albumin standards.
Specific IgG and IgA antibodies developed against the CWH of LGG were titrated in all the sera. Two
Homology searches using the CWH amino acid sequence (gi|258507319; ref|YP_003170070.1) were performed using the BlastP algorithm implemented at the National Center for Biotechnology Information (
Antibodies developed against the CWHp and present in the following sera C72, C65, C49, C59, P8, P39, P34, and P20 were determined by ELISA as described before. These sera were chosen as being representative for the specific CWH anti-IgA/anti-IgG values. Five
In order to evaluate and analyze distribution of data, the following tests were applied: the Runs test, Levène’s test, and the Kolmogorov-Smirnov test. The nonparametric Kruskal-Wallis test was used for median comparison between groups (healthy controls, CD, UC, and IBD). All tests were performed using SPSS v18.0 software.
Several immunological studies have shown that the human immune system, in the framework of IBD, reacts differentially against many of the microorganisms composing our gut microbiota by producing specific IgA and IgG responses such as ASCA [
Susceptibility to IBD has been associated with the so-called leaky gut syndrome (LGS), in which a decrease in the gut epithelial barrier led to an increase of antigens from food and bacteria from the gut lumen leaking into the body [
Extracellular proteins from the bacteria strains used in this study, all of them representative of microorganisms used as probiotics or in human nutrition, were immobilized on PVDF membranes and submitted to immunoblot analysis. Incubation with pools of sera of the three different groups (HC, CD, and UC), showed the presence of different immunoreactive bands when membranes where developed using an anti-human IgA antibody conjugated with horseradish peroxidase (Figure
(a) Immunoreactive bands corresponding to antibodies present in the sera of healthy controls or IBD patients raised against the extracellular proteins of the bacterial strains used in this study. (b) Western blotting analysis showing different patterns of sera immunoreactivity against extracellular proteins produced by the strains used in this study (C# healthy controls; P# IBD patients). In all cases, a secondary anti-human IgA antibody, HRP conjugated, was used. The order of the lanes (lines 1 to 6) correspond to that of the SDS-PAGE.
At this point, our results strongly suggested that the levels of specific anti-CWH IgAs were increased in the IBD sera. As Western-blot is very limited for antibody quantification, we purified enough amounts of CWH following the procedure described in the Material and Methods section. Once CWH was purified, and after verifying its purity by SDS-PAGE, the protein was used for the coating of 96-well plates and for quantifying the levels of specific antibodies present in the sera collection. After incubation of the different sera with the coated CWH, we used two types of secondary antibodies for detection of different antibody isotypes specifically developed against CWH, anti-human IgG, and anti-human IgA.
While levels of specific IgGs remained constant between the two populations (healthy controls versus IBD patients) with the exception of some outlier, levels of anti-CWH IgA were significantly higher in the IBD sera (Figure
Distribution of the specific anti-CWH titers (IgG and IgA) of all the sera divided into two groups (HC: healthy controls, IBD: inflammatory bowel disease) (
Distribution of the specific anti-CWH titers (IgG and IgA) of all the sera assayed taking into account three groups: healthy controls (HC), Crohn’s disease (CD), and ulcerative colitis (UC).
In a step forward, we compared the amino acidic sequence of the CWH protein from LGG against the nonredundant protein database at the NCBI servers, in order to determine whether the anti-CWH antibodies were or were not specific to this protein. A BLAST analysis allowed us to identify a 13-mer peptide which was only present in the CWH from
Comparison between the titers values obtained in different sera (C#: healthy controls, P#: IBD patients) using different target proteins (CWH: purified cell wall hydrolase, CWHp: specific CWH peptide) and different secondary antibodies (Anti-human IgA and IgG). Results are expressed as the median of 3 independent measures.
Patient | Anti CWHp-IgG (antiCWH IgG) | Anti CWHp-IgA (antiCWH IgA) |
---|---|---|
C72 | 0 (2,500) | 250 (2,500) |
C65 | 0 (500) | 500 (1,000) |
P34 | 0 (2,500) | 250 (2,500) |
P20 | 0 (500) | 1,000 (10,000) |
C49 | 0 (250) | 500 (1,000) |
C59 | 0 (250) | 500 (1,000) |
P39 | 0 (500) | 500 (2,500) |
P8 | 0 (500) | 1,000 (2,500) |
Surprisingly, titers corresponding to the specific CWH-peptide IgG antibodies were zero in all the sera, suggesting that they have been developed against homologous surface proteins of other Gram positive bacteria. In this sense, presence of certain enteropathogens, notably infection by
Why levels of IgA antibodies against extracellular proteins produced by gut bacteria are more elevated in IBD patients? It is known that increased gut permeability may correlate with IBD susceptibility/risk. This elevated passage of bacterial antigens in the intestinal mucosa may lead to a concomitant increase in the immune response through specific antibody production for compensating this failure in the barrier function of the gut epithelium [
Our experiments showed that IgA antibodies specifically developed against an extracellular protein of
The authors declare that there is no conflict of interests regarding the publication of this paper.
Borja Sánchez and Arancha Hevia were recipients of a Ramón y Cajal postdoctoral contract and a FPI Grant, respectively, from the Spanish Ministry of Economy and Competitiveness. Research in our group is supported by Grants AGL2010-14952 and RM2010-00012-00-00 from the Spanish Plan Nacional de I+D.