42NISR Research GRANT
2007 Research Grant

Development of molecular immunoevaluation system forprobiotics
Haruki KITAZAWA
Division of bioscience and biotechnology for future bioindustries, Graduate School of Agricultural Science, Tohoku University(HEK) 293 cells were transfected with the mammalian expression vectors encoding porcine PRR by lipofection
method (1–3). Expression of porcine PRR was confirmedby reverse transcription-polymerase chain reaction, flow
cytometry and confocal laser microscopy (1–3). The nuclear factor-k B (NF-k B) reporter assay was performed asdescribed elsewhere (1–3). Briefly, the porcine PRR-expressing transfectants were transfected with pGLM-ENHluci vector, which encodes the human NF-k B gene A1 andA2 sites. After stimulation with the indicated stimulants, luciferase assays were performed according to the manufacturer’s protocol (Promega, Tokyo, Japan).

Research aims
There is currently growing interest in the worldwide
studies on physiologically functional foods and feeds that
can improve human and animal health. In Japan, the Foodfor Specified Health Uses, so-called FOSHU, have been authorized by the Ministry of Health, Labor and Welfare since1993. To date, around 800 products have been approved as
FOSHU in several categories. Among them, particularly
many kinds of fermented products containing probiotics,
which are expected to improve human intestinal microbialbalance, are widely available in the market. However, immunologically functional foods containing probiotics havenot been approved as FOSHU due to the lack of a unified
evaluation standard for immunological activity even if there
were many reports suggesting their immunological effects.
In this study, we tried to develop a certain molecular immunoevaluation system for immunobiotics derived from probiotics. For this purpose, we established an evaluation systemusing the pattern recognition receptors (PRRs), which are
known to play an essential role in innate immunity. In particular, we used porcine PRR instead of mouse PRR, sinceswine is phylogenetically more close to human than mice.

Results
In the present study, cDNAs encoding porcine nucleotide-binding oligomerization domain-1 (NOD1), NOD2,and radioprotective 105 (RP105)/MD-1 were isolated fromporcine GALT (Table 1). Comparison of the ORF sequences of porcine PRRs with those of human and mousecounterparts indicated that porcine PRRs are more similar
to human than to mouse (Table 1).
In order to develop the molecular immunoevaluation system for immunobiotics, we constructed porcine PRR-overexpressing HEK 293 cells. In the expression analysis, weconfirmed the expression of PRR in the transfectants at thenucleotide and protein levels. Using the porcine NOD-expressing transfectans, we evaluated the immunological response to low-molecular peptidoglycan (PGN) fragmentsthat are essential components of gram-positive bacteriasuch as immunobiotic lactic acid bacteria. When stimulated
by muramyldipeptide (MDP; N-acetylmuramyl-L-alanyl-Disoglutamine, MurNAc-L-Ala-D-isoGln), there was a significant increase in NF-k B dependent luciferase activity in theNOD2-expressing transfectans (Fig. 1). In the analysis ofMethods
The complementary DNAs (cDNAs) encoding porcine
PRR were cloned from gut-associated lymphoid tissues(GALT) of adult swine (1–3). The first-stranded cDNAs
were synthesized from the total RNA from porcine GALT
by using the primers whose sequences were designed frommultiple alignments of the conserved sequences of humanand mouse counterparts. The PCR products containing thecomplete open reading frames of porcine PRR were sequenced and characterized. Human embryonic kidney
Table 1.

Characterization of porcine PRRs (NOD1, NOD2, RP105 and MD-1)

PRRs

Length of ORF

Homology to human

Homology to mouseAccession

NOD1
NOD2
RP105
MD-1

nu*1 (bp)aa*2nu (%)

aa (%)

nu (%)

aa (%)

2862
3039
1986
480953
1013
661
159

86.7
84.4
84.1
77.7

83.8
81.6
77.676.5

80.9
77.9
75.3
72.8

79.2
76.6
7071.1*1 nunucleotides, *2 aaamino acids

AB187219
AB195466
AB190767
AB190766

43Fig. 1. Schematic representation of the recognition of immunobiotics
and immunogenics by NOD1, NOD2 and RP105/MD-1.

NOD1-expressing transfectans, NF-k B was significantly
activated by the stimulation with g -D-glutamyl-meso-diaminopimelic acid (iE-DAP). Furthermore, we observedthat meso-diaminopimelic acid (meso-DAP) has an ability
to induce the immune activation via NOD1-mediated signaling pathway (Fig. 1).
We have reported that the extracellular phosphopolysaccharides (EPS) produced by Lactococcus lactis subsp. cremoris (Lc. cremoris) exerts immunological functions suchas antitumor and immunostimulatory activities (4,5). Because the precise mechanisms by which the PRR contributeto the recognition of EPS have not been elucidated, we examined the involvement of RP105/MD-1 in cellular immune activation by EPS. In the NF-k B-dependent lucifarase assay, EPS from Lc. cremoris strongly induced immune activation via RP105/MD-1 (Fig. 1). In addition, weobserved that RP105/MD-1-mediated immune activaiton
was involved in phosphatidylinositol 3-kinase and Bruton’styrosine kinase pathway (Fig. 1). The results suggest thatEPS from Lc. cremoris is a potent immunoregulator recognized by RP105/MD-1 complex.

In this study, we constructed the porcine NOD1-, NOD2-,or RP105/MD-1-expressing transfectants, and identified
some immunoregulatory factors as novel immunogenics on
the basis of the relationship between PRRs and their ligands. In our previous studies, we have cloned several
porcine Toll-like receptor family members and analyzed thespecificity of their ligands from immunobiotic lactic acidbacteria (8–13). Together with our previous and present
studies, we successfully developed various kinds of porcine
PRR-expressing transfectants as novel molecular immunoevaluation system, which are useful for understanding theimmunoregulatory effects of individual immunogenics, andscreening high potential immunobiotic lactic acid bacteria
strains via PRR signaling. Our system may also provide important findings at the molecular level on PRR signal transduction pathways and recognition mechanisms for the specific ligands. Screening of immunobiotic lactic acid bacteria strains with certain immunogenics, and better understanding of intestinal immunoregulation should help in thedevelopment of not only immunologically functional foods
but also oral vaccines that specifically target intestinal immune responses.
References
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Conclusion

6)

Studies on the immunological functions of immunobiotic
lactic acid bacteria and immunogenics have contributed totheir worldwide application as functional foods, supplements and feeds (6,7). With respect to the health-promoting
effects of immunobiotics, we believe that several structural
factors of these immunobiotics are recognized by PRRs on
innate immune cells in the GALT. In the present study,
porcine PRRs were shown to be more close to human thanmouse counterparts, supporting the notion that swine may
be a good model for human intestinal immune system ascompared with mouse. The clarification of the immune system mediated by porcine PRRs in the swine GALT is important to develop a model system for studies on humanmucosal immune system.

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