How does H. pylori inhibit the immune responses that specific to it? As
mentioned in introduction, many reports suggested that virulence factors may play the role to suppress the host immune system. However, these factors all have their own defects to help every strain of H. pylori survive and escape from the immune response [21-25, 28, 30, 50]. Thus, we thought there are some factor else that helps H. pylori inhibit immune responses needs to coincide with three features: i) it presents in every stain, ii) its amino acid sequence is mostly conserved in every strain, and iii) it should exhibit immune suppressive ability. After checking the eight common virulence factors with these three features, Hp hsp60 has the highest homology and is present in every strain. On the other hand, Hp hsp60 also helps H. pylori to adhere and colonize in gastric mucosa [40], which means this protein has posibility to contact with
immune cells. Taken these together, Hp hsp60 has the highest possibility to help H.
pylori as long as it has the ability to suppress immune responses.
In this study, we found that after treated with Hp hsp60, the cell proliferation of PBMC was seriously inhibited and Jurkat cells were slightly inhibited. However, the cell proliferation of CD3+ T cell was not affected. This proliferation inhibition might be due to the cell arrest. Besides, the CD4+CD25+ T cells percentage in Hp hsp60
treated PBMC and CD3+ T cells were both increase. These increased CD4+CD25+ T cells were confirmed as Treg by detecting the induction of foxp3 mRNA expression.
Taking these finding together, we found that Hp hsp60 has the ability to help H. pylori escape from the immune attacks by increasing the number of Treg which inhibit the proliferation of PBMC. However, there were also some doubtful points in our study.
These controversial results between PBMC and CD3+ T cells might be due to whether the cells had been activated. Because the monoclonal antibody we used in T cell isolation has the ability to activate the T cells. During the T cell purification procession, this antibody might have activated the T cells. This activation can be confirmed by the difference between cells that cultured with or without antibody. The difference in PBMC is about 40%, however, the difference in CD3+ T cells only 17%.
Therefore, when cells treated with Hp hsp60, PBMC was inactivated, whereas CD3+ T cells had been activated. Thus, whether the cells have been activated might be the key factor that influences the inhibitory function of Hp hsp60.
In our study, we detected the increased number of Treg after treated with Hp hsp60, but where did the Treg come from? According to the literatures, CD4+CD25+ Treg are arisen from two major sources. Natural CD4+CD25+ Treg were generated in
the thymus and the proliferation of nTreg can be stimulated by antigen stimulation [51, 52]. On the contrary, the adaptive CD4+CD25+ Treg were converted from CD4+CD25 -T cells in the peripheral blood under the help of -TGF-β and IL-2 [53]. If the
proliferated Treg were arisen from nTreg, they needed the help of antigen presentation.
Although the APCs in PBMC could give this help, however, there were no any other APCs in CD3+ T cells. Thus, these enhancements might not be associated with nTreg in the CD3+ T cell culture system. If the proliferated Treg were converted from CD4+CD25- T cells, it also needed the help of TGF-β. Therefore, if Hp hsp60 could stimulate the secretion of TGF-β from PBMC and CD3+ T cells, Hp hsp60 might influence the differentiation of Treg though this mechanism. In our preliminary data, we also test the TGF-β1 level in the culture supernatant of PBMC and CD3+ T cells after treated with Hp hsp60 (Figure 9). In this experiment, we used the ELISA kit purchased from Promega. Although the TGF-β1 concentration were increase in a dose-dependent curve, unfortunately, we also found that this ELISA kit cross reacted with Hp hsp60 recently. The relationship among Hp hsp60, Treg, and TGF-β is still not clear. Thus, we could not provide strong enough evidence about the source of these increased Treg.
There might be some other ways to increase percentage of Treg that may help H.
pylori escape from immune system. The escape mechanisms might be similar to that
of Mt hsp60. In human T cell differentiation progress, the negative and positive selections delete almost self-reactive T cells. However, minor self-reactive T cells would escape from these selections and play a role in immune suppression by acting as regulatory T cells. Self-hsp60-specific CD4+ T cells might be activated in many ways. First, because of the high homology of amino acid sequence, the self-hsp60 specific CD4+ T cells might also be activated by cross reaction after exposed with Hp hsp60 in the mucosa-associated lymphoid tissue. The crossreactive T cells might be maintained by non-professional antigen-presenting cells such as gastric epithelial cells and differentiated to regulatory T cells [54]. Second, after infected with H. pylori, human hsp60, which acts as a danger signal, might be released from damage cells and then activated the self-hsp60 specific CD4+ T cells. On the other hand, the secreted human hsp60 might decrease the function of Hp hsp60-specific T cells by
altered-peptide- ligand (APL) effect. The APL is defined as analog of immunogenic peptide in which the TCR contact sites have been manipulated. These high homology peptides still reacts with T cells but only partially activate T cells. This partial
activation often not includes the cell proliferation. Taken these mechanisms, H. pylori can escape from the immune system easily.
Figuure1
Figuree
Figure1. The PCR product and the cloning RE check gel electrophoresis. All DNA were checked with 0.8% agarose gel. (A) The PCR product should be 1641bps and the marker pointed by arrow is 1.5kb. (B) The vector and insert were both digested with EcoR I and Xho I. The samples were loaded in the order: M: 1kb marker, 1: uncut vector, 2: vector + EcoR I + Xho I (5422bps), 3: uncut insert, 4:
insert + EcoR I + Xho I (1641bps). (C) The plasmids isolated from ligation results were screened with RE check. The samples were loaded with the follow order: M:
1kb marker, 1: uncut plasmid, 2~4: plasmid + EcoR I + Xho I. The digested fragments should be 5422 and 1641bps. The spots pointed by arrow might be RNA contaminant.
Figure 2
Fraction number Fraction number
Figure 2. The protein concentration of each fraction in the protein purify procession. We collected each fraction with the 1ml volume during the purify
procession which were consisted of (A) wash, (B) elution, and (C) desalting. 10 μl of sample from each fraction were reacted with 290 μl coomassie reagent at room temperature in the dark for 10 min and followed measured by OD 595nm. Each bar represented the mean value ± SD (standard deviation).
Fraction number
Figu
Figure 4
Figure 4. The PBMC and CD3+ T cell purity. (A)PBMC was purified human whole blood by Fique Plus and detected by flow cytometry with FSC and SSC channels. The lymphocytes, monocytes, and granulocytes were pointed by red, blue, and purple arrows, respectively. (B) CD3+ T cells were purified by magnetic bead. To test the purity, the CD3+ T cells were stained with (blue line) and without (black line) anti-human CD3-FITC respectively and detected with FL1 channel. The average of the cell purity is over 99%.
>199 %
Figure 5
Figure 5. The effect of Hp hsp60 on proliferation of PBMC. Hp Hsp60 added to a concentration of 1, 5, 10 μg/ml (black wedges) to treat with PBMC (2*105cells/well) for 4 days. The PBMC were seeded in the anti-human CD3 mAb pre-coaded 24 well plate. The relative absorption at 490nm were calculated with the followed equation:
the relative OD490 = the OD490/ the average of OD490 of the control. The value of anti-CD3 mAb unstimulated group was 0.591 ± 0.040. The value of anti-CD3 mAb stimulated but Hp hsp60 untreated group was 1.000 ± 0.123. The values of anti-CD3 mAb stimulated cell treated with different dose of Hp hsp60 were 0.697 ± 0/154, 0.705 ± 0.121, and 0.791 ± 0.125. Each bar represented the mean value ± SD from two independent experiments. (*: P < 0.05 indicated a significant difference compared to the control group. **: P < 0.005 indicated a significant difference compared to the control group.)
Figure 6
Figure 6. The effect of Hp hsp60 on proliferation of CD3+ T cells. CD3+ T cells (2*105 cells/well) were treated with 10 μg/ml of Hp Hsp60 for 4 days. The CD3+ T cells were seeded in the anti-human CD3 mAb pre-coaded 24 well plate. The relative absorption at 490nm were calculated with the followed equation: the relative OD490
= the OD490/ the average of OD490 of the control. The value of anti-CD3 mAb unstimulated group was 0.844 ± 0.061. The value of anti-CD3 mAb treated but Hp hsp60 untreated group was 1.000 ± 0.032. The value of anti-CD3 mAb stimulated and Hp hsp60 treated group was 1.048 ± 0.062. Each bar represented the mean value ± SD from three independent experiments.
Figure 7
Figure 7. The effect of Hp hsp60 on proliferation of Jurkat cells. Hp Hsp60 added to a concentration of 1, 5, and 10 μg/ml (black wedges) to treat with Jurkat cells (2*104cells/well) for 2 days. The Jurkat cells were seeded in the anti-human CD3 mAb pre-coaded 24 well plate. The relative absorption at 490nm were calculated with the followed equation: the relative OD490 = the OD490/ the average of OD490 of the control. The value of the untreated group was 1.000 ± 0.017. The values of Jurkat cells treated with different dose of Hp hsp60 were 1.026 ± 0.043, 1.042 ± 0.083, and 0.958 ± 0.019, respectively. Each bar represented the mean value ± SD from three independent experiments. (**: P < 0.005 indicated a significant difference compared to the control group.)
Figure 8
Figure 8. Different physiological stage of cells in Annex V-FITC / PI double staining assay. In our experiment, we used Annexin V-FITC /PI double staining assay to determine the physiological stage of cells. The vertical and cross axle mean the fluorescence intensity of Annexin V-FITC and PI respectively. The cells were cultured with or without anti-CD3 mAb, respectively. The anti-CD3 mAb stimulated cells also were divided into two groups that treated with or without Hp hsp60. After detected with flow cytometry by FL1 and FL3 channels, the results were presented in dot plots with quadrant line. The lower right part was defined as the apoptotic cells (the blue frame). The sum of the up left and up right part were defined as the necrosis cells (the orange frame). The lower left part was defined as the alive cells (the red frame).
Figure 9
Figure 9. The effect of Hp hsp60 on PBMC cell proliferation inhibition. PBMC were cultured with the same condition of the cell proliferation experiments. After stained with Annexin V-FITC / PI, the cells were detected with flow cytometry by FL1 and FL3 and the cell percentage were calculated as shown in Figure 8. The apoptosis cell percentage of different groups were list as shown in the picture, 4.943 ± 8.497, 2.139 ± 3.189, 1.548 ± 1.364, 2.384 ± 2.926, and 4.144 ± 5.080, respectively.
The necrosis cell percentage of different groups were 23.488 ± 3.190, 26.239 ± 10.004, 29.583 ± 9.620, 31.744 ± 8.053, and 32.168 ± 8.092, respectively. The alive cell percentage of different groups were 71.630 ±6.633, 71.623 ± 10.424, 68.870
±10.904, 68.870 ± 10.904, 65.841 ± 9.082, and 63.690 ± 11.134. Each bar represents the mean value ± SD from four independent experiments.
Figure 10
Figure 10. The effect of Hp hsp60 on CD3+ T cell physiology. CD3+ T cells were cultured with the same condition of the cell proliferation experiments. After stained with Annexin V-FITC / PI, the cells were detected with flow cytometry by FL1 and FL3. The cell percentages were calculated as shown in Figure 8. The apoptosis cell percentage of different groups were list as shown in the picture, 1.11 ± 0.86, 1.29 ± 1.51, and 0.55 ± 0.43, respectively. The necrosis cell percentage of different groups were 23.63 ± 9.87, 26.34 ± 9.62, and 27.36 ± 12.02, respectively. The alive cell percentage were 75.10 ± 9.46, 72.37 ± 9.37, and 72.10 ± 12.03, respectively. Each bar represents the mean value ± SD from six independent experiments.
Figure 11
Figure 11. The CD4+CD25+ T cell percentage in CD4+ T cells. In our experiment, we used anti-human CD4-FITC/ anti-human CD25-PE double staining assay to determine the CD4+CD25+ T cell percentage in CD4+ T cells. The vertical and cross axle mean the fluorescence intensity of anti-human CD4-FITC and anti-human CD25-PE, respectively. The cells were cultured with or without anti-CD3 mAb, respectively. The anti-CD3 mAb stimulated or unstimulated cells also were divided into two groups that treated with or without Hp hsp60. The cells were harvested and stained with anti-human CD4-FITC and anti-human CD25-PE. The cell fluorescence was detected by FL1 and FL2 channels with compensation, the results were presented in dot plots with quadrant line. The sum of lower right and up right parts were defined as CD4+ T cells (the blue frame). The up right part was defined as CD4+CD25+ T cells (the red frame). The cell percentage = the red frame/ the blue frame.
Figure 12
Figure 12. The effect of Hp hsp60 on CD4+CD25+ T cell population of CD4+ T cells in PBMC. 106 PBMC were divided into two groups to culture with or without anti-CD3 mAb. These groups were treated with or without Hp hsp60 for 6 days. The equation of CD4+CD25+ T cell percentage in CD4+ T cells has shown in Figure 11.
The triangles mean the samples we had been test. Mean values are indicated by horizontal bars. The percentage of the anti-CD3 mAb unstimulated and Hp hsp60 untreated group was 36.26 ± 10.09 %. The percentage of the anti-CD3 mAb
unstimulated but Hp hsp60 treated group was 48.21 ± 9.11 %. The percentage of the anti-CD3 mAb stimulated but Hp hsp60 untreated group was 64.69 ± 1.89 %. The percentage of the anti-CD3 mAb stimulated and Hp hsp60treated group was 70.98 ± 3.34 %. These samples were from four different donors. (*: P < 0.05 indicated a significant difference compared to the untreated group.)
Figure 13
Figure 13. The effect of Hp hsp60 on CD4+CD25+ T cell population of CD4+ T cells in CD3+ T cells. The CD3+ T cells were treated with different culture conditions as shown in the figure for 6 days. The cells were harvested and stained with
anti-human CD4-FITC and anti-human CD25-PE. The fluorescence was detected by FL1 and FL2 channels with compensation. The results were presented in dot plots with quadrant lines as shown in Figure 11. To calculate the relative CD4+CD25+ T cell percentage in CD4+ T cell, cell percentages were calculated with the equation: the cell percentage = the up right / (up right + lower right). In Figure 13B, the relative
CD4+CD25+ T cell percentage in CD4+ T cells was calculated with the equation: the relative cell percentage = sample cell percentage / control cell percentage. Each bar represented the mean value ± SD from multiple independent experiments. (*: P < 0.05 indicated a significant difference compared to the untreated group.)
Figure 14.
Figure 14. The foxp3 mRNA expression in T cells. After cultured with the same condition in flow cytometry experiment, mRNA were isolated from 106 CD3+ T cells and then reverse transcripted into cDNA. The foxp3 mRNA expression levels were detected by real-time PCR. The β-actin was used as loading control. Each bar represented the mean value from one independent experiment.
Figure 15
Figure 15. The TGF-β1 concentration in Hp hsp60-treated PBMC and CD3+ T cell supernatant. (A) 106/ml PBMC were treated with different doses of Hp hsp60 (100 pg/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1 μg/ml, 10 μg/ml, and 30 μg/ml) in 24 well plate for 24 hr. (B) 106 CD3+ T cells were also treated with 10 μg Hp hsp60 and boiled Hp hsp60 for 24 hr. The culture supernatant were harvested and the TGF-β1
concentration were detected by ELISA kit (Promega).
Table
Lower immunogenic ability Gewirtz, A.T., J Infect Dis , 2004
HP‐NAP neutrophil Modulate the oxidative burst Med Microbiol Immunol., Petersson C, 2006
CagA B cells Growth inhibition Umehara, S., Oncogene, 2003
T cells Reduce IL‐4 mRNA expression Orsini, B., Infect Immun, 2003
VacA B cells Inhibit the processing of
T cells Inhibit the IL‐2 expression Gebert, B., Science, 2003
T cells IL‐2Rα surface location Gebert, B., Science, 2003
T cells Inhibit T cell proliferation Boncristiano, M., J Exp Med, 2003
PBL Reduce phosphorylation and
activation of Rb and cell arrest
Gebert, B., Science, 2003
Table 1. The reference about the effects of H. pylori virulence factors on immune suppression. In this table, we collected the reports about the inhibition functions of Arginase, Flagellin, HP-NAP, CagA and VacA. The relationship between these factors and immune responses were investigated. NOS: Nitrous Oxide Synthase. PBL:
Peripheral Blood Lymphocyte.
Table 2
Protein name Amino acid sequence length
Table 2. The homology of H. pylori virulence factors among different strains.
These eight virulence factors are arranged in the order of the percentage of identity.
Percent of identity means the percentage of the identical amino acids in the full length.
Percent of positive means the percentage of the same group amino acids in the full length. The alignments were done by comparing the sequences from at least four stains. The alignment results can be seen in Appendix. The strains compared in this table were listed as follow: Hsp60: 26695, HC28 (used in our study), CCUG 178874, HPAG1, and J99. Hsp70: 26695, HPAG1, J99, Shi470. UreB: Ch-CTX1, Iran-HP031, J99, HC28, Y06, MEL-HP27. Arginase: 26695, G27, AG1, B5, B7, and J99. SabA:
G1, M30, M23, G26, and M65. BabA: 92-18, 92-26, J116, CCUG 17875. VacA:
ATCC 49503, J99, F26, OK111, NCTC11638, and TX30A. CagA: ATCC43526, F26, J16, J99, NCTC11638, and OK111.
Table 3
Disease model Hsp Species Reference
Arthritis Hsp10 M. tuberculosis [55]
Arthritis Hsp60 M. tuberculosis [56]
Arthritis Hsp70 M. tuberculosis [57]
Arthritis GroEL E. coli [58]
Arthritis DnaK E. coli [58]
Type 1 diabetes Hsp60 M. tuberculosis [59]
Type 1 diabetes Hsp60 – derived peptide
Homo sapeins [60]
Atherosclerosis Hsp60 M. tuberculosis [61]
Experimental allergic encephalomyelitis
Hsp60 M. tuberculosis [62]
Allergic asthma Hsp60 M. leprae [63]
Table 3. The immune suppression of different species Hsps protect the host from different disease models. These disease models were all experimental inflammatory diseases that have been proven to be rescued by the administration of Hsps. GroEL means the hsp60 of E. coli. DnaK means the hsp70 of E. coli.
Appendix
Appendix 1
1. The amino acid homology of different virulence factors in different stains.
1.1 Hsp60
1.1.1 Alignment result
1.1.2 Strains: 26695, HC28 (used in our study), CCUG 178874, HPAG1, and J99.
1.1.3 Identity: 99.1%
1.1.4 Positive: 100%
1.2 Hsp70
1.2.1 Alignment result
1.2.
1.2.
1.2.
1.3 1.3.
2 Strains:
3 Identity 4 Positive UreB 1 Alignm
: 26695, HP y: 98.1%
e: 100%
ment result:
PAG1, J99, SShi470
1.3.2 Strains: Ch-CTX1, Iran-HP031, J99, HC28, Y06, MEL-HP27.
1.3.3 Identity: 95.6%
1.3.4 Positive: 100%
1.4 Arginase
1.4.1 Alignment result:
1.4.2 Strains: 26695, G27, AG1, B5,B7, and J99
1.4.3 Identity: 91%
1.4.4 Positive: 100%
1.5 SabA
1.5.1 Alignment result:
1.5.2 Strains: G1, M30, M23, G26, and M65 1.5.3 Identity: 78.6%
1.5.4 Positive: 97.1%
1.6 BabA
1.6.1 Alignment result:
1.6.2 Strains: 92-18, 92-26, J116, CCUG 17875 1.6.3 Identity: 76.4%
1.6.4 Positive: 96.7 1.7 VacA
1.7.1 Alignment result:
1.7.2 Strains: ATCC 49503, J99, F26, OK111, NCTC11638, and TX30A 1.7.3 Identity: 68.9%
1.7.4 Positive: 96.6%
1.8 CagA
1.8.1 Alignment result:
1.8.
1.8.
1.8.
2 Strains:
3 Identity 4 Positive
: ATCC4352 y: 62.9%
e: 93.7%
26, F26, J166, J99, NCTTC11638, annd OK111
2. pET-Hp hsp60 2.1 Map
2.2 H. pylori hsp60 DNA sequence
1 atggcaaaag aaatcaaatt ttcagatagt gcaagaaacc ttttatttga aggcgtgaga 61 caactccatg acgctgtcaa agtaaccatg gggccaagag gtaggaatgt gttgatccaa 121 aaaagctatg gcgctccaag catcaccaaa gatggcgtga gcgtggctaa agagattgaa 181 ttaagttgcc cggtagctaa catgggcgct caactcgtta aagaagtagc gagcaaaacc 241 gctgatgctg ccggcgatgg cacgaccaca gcgaccgtgc ttgcttatag catctttaaa 301 gaaggcttga ggaatatcac ggctggggct aaccctattg aagtgaaacg aggcatggat 361 aaagccgctg aagccattat taatgagctt aaaaaagcga gcaaaaaagt aggtggtaaa 421 gaagaaatca cccaagtagc gaccatttct gcaaactccg atcacaatat cgggaaactc 481 atcgctgacg ctatggaaaa agtgggtaaa gacggcgtga tcaccgttga agaagctaag 541 ggcattgaag atgaattaga tgtcgtagag ggcatgcaat ttgatagagg ctacctctcc 601 ccttactttg taacaaacgc tgagaaaatg accgctcaat tggataacgc ttacatcctt 661 ttaacggata aaaaaatctc tagcatgaaa gacattctcc cgctactaga aaaaaccatg 721 aaagagggca aaccgctttt aatcatcgct gaagacattg agggcgaagc tttaacgact 781 ctagtggtga ataaattaag aggcgtgttg aatatcgcag cggttaaagc tccaggcttt 841 ggggacagaa gaaaagaaat gctcaaagac atcgctgttt taaccggcgg tcaagtcatt 901 agcgaagaat taggcttgac tttagaaaac gctgaagtgg agtttttagg caaagccgga 961 aggattgtga ttgacaaaga caacaccacg atcgtagatg gcaaaggaca tagccatgat 1021 gttaaagaca gagtcgcgca aatcaaaacc caaattgcaa gcacgacaag cgattatgac
pET-Hp hsp60
1081 aaagaaaaat tgcaagaaag attggccaaa ctctctggtg gtgtggctgt gattaaagtg 1141 ggcgctgcga gtgaagtgga aatgaaagag aaaaaagacc gggttgatga cgcattgagt 1201 gcgactaaag cagctgttga agagggcatt gttattggcg gcggtgcggc tctcattcgc 1261 gcggctcaaa aagtgcattt gaatttacac gatgatgaaa aagtaggcta tgaaatcatc 1321 atgcgtgcca ttaaagcccc attagctcaa atcgctatca atgccggtta tgatggcggt 1381 gtggtcgtga atgaagtgca aaaacacgaa gggcattttg gttttaacgc tagcaatggc 1441 aagtatgtgg atatgtttaa agaaggcatt attgacccct taaaagtaga aaggatcgct 1501 ttacaaaatg cggtttcggt ttcaagcctg cttttaacca cagaagccac cgtgcatgaa 1561 atcaaagaag aaaaagcaac cccagcaatg cctgatatgg gtggcatggg cggtatggga 1621 ggcatgggcg gcatgatgta a
2.3 H. pylori hsp60 protein sequence
1 makeikfsds arnllfegvr qlhdavkvtm gprgrnvliq ksygapsitk dgvsvakeie 61 lscpvanmga qlvkevaskt adaagdgttt atvlaysifk eglrnitaga npievkrgmd 121 kaaeaiinel kkaskkvggk eeitqvatis ansdhnigkl iadamekvgk dgvitveeak 181 giedeldvve gmqfdrgyls pyfvtnaekm taqldnayil ltdkkissmk dilpllektm 241 kegkplliia ediegealtt lvvnklrgvl niaavkapgf gdrrkemlkd iavltggqvi
1 makeikfsds arnllfegvr qlhdavkvtm gprgrnvliq ksygapsitk dgvsvakeie 61 lscpvanmga qlvkevaskt adaagdgttt atvlaysifk eglrnitaga npievkrgmd 121 kaaeaiinel kkaskkvggk eeitqvatis ansdhnigkl iadamekvgk dgvitveeak 181 giedeldvve gmqfdrgyls pyfvtnaekm taqldnayil ltdkkissmk dilpllektm 241 kegkplliia ediegealtt lvvnklrgvl niaavkapgf gdrrkemlkd iavltggqvi