ARTICLE ADDENDUM
Gut Microbes 1:5, 325-329; September/October 2010; © 2010 Landes Bioscience
Helicobacter pylori directs tolerogenic programming of dendritic cells
Min Zhang, Maochang Liu, Jay Luther and John Y. Kao*
Department of Internal Medicine; Division of Gastroenterology; University of Michigan Health System; Ann Arbor, MI USA
Key words: regulatory T cells, Th17,
immune escape, tolerance, two-photon
microscopy
Abbreviations: AL-DC, dendritic cells
pulsed with Acinetobacter lwoffii; DC,
dendritic cell; EC-DC, dendritic cells
pulsed with Escherichia coli; HP-DC,
dendritic cells pulsed with Helicobacter
pylori; IL, interleukin; IFN, interferon;
PBS-DC, dendritic cells pulsed with
phosphate-buffered saline; PCR, poly-
merase chain reaction; TGF, transform-
ing growth factor; Th17 cell, IL-17+
helper T cell; Treg, regulatory T cell
Submitted: 04/27/10
Revised: 07/15/10
Accepted: 07/16/10
Previously published online:
www.landesbioscience.com/journals/
gutmicrobes/article/13052
DOI: 10.4161/gmic.1.5.13052
*Correspondence to:
John Y. Kao; Email: jykao@umich.edu
Addendum to: Kao JY, Zhang M, Miller MJ,
Mills JC, Wang B, Liu M, et al. Helicobacter pylori
immune escape is mediated by dendritic cell-
induced Treg skewing and Th17 suppression in
mice. Gastroenterology 2010; 138:1046–54.
www.landesbioscience.com Gut Microbes 325
O ur laboratory has shown that
Helicobacter pylori infection in
mice triggers an increase in the number
of subepithelial lamina propria CD11c +
dendritic cells with luminal projections.
The physical characteristic of these cells
is consistent with their ability to traverse
epithelial tight junctions as reported by
Maria Recigno.3 Gastric CD103 + den-
dritic cells, which are known to induce
mucosal regulatory T cells, were also
increased in number, raising the ques-
tion whether H. pylori infection induces
a regulatory T cell-skewed response by
way of a bacteria-dendritic cell inter-
action. In fact, bone marrow-derived
dendritic cells underwent tolerogenic
programming, skewing the balance
between effector and regulatory T cell
responses towards regulatory T cell dif-
ferentiation in a transforming growth
factor-β- and interleukin-10-depen-
dent manner. Depletion of regulatory
T cell numbers augmented H. pylori-
specific effector helper T cell responses,
which correlated with a lower degree of
H. pylori colonization. These results sug-
gest H. pylori is capable of inducing a
regulatory T cell-skewed response that
limits the host’s ability to eradicate the
bacteria, allowing the H. pylori infec-
tion to persist. To better understand
the mechanism of H. pylori tolerogenic
programming we compared the differ-
ential expressions of 34 genes critical for
dendritic cell function in bone marrow-
derived dendritic cells pulsed with live
H. pylori or other Gram-negative bacte-
ria (e.g., Escherichia coli, Acinetobacter
lwoffii). Our data imply that H. pylori
targets the Toll-like receptor 2 pathway
to induce a regulatory T cell-skewed
response. In addition, we show that H.
ARTICLE ADDENDUM
pylori-pulsed dendritic cells are capable
of inducing the conversion of naïve T
cells to regulatory T cells. These observa-
tions are evidence of a unique tolerogenic
program in dendritic cells that involves
active editing of the immune response by
H. pylori, favoring its persistence in the
gastric mucosa.
Helicobacter pylori-Stimulated
Dendritic Cells Induce Regulatory
T Cell Skewing
Dendritic cells (DCs), first described in
1973,1 are professional antigen-presenting
cells, capable of inducing antigen-specific
adaptive immune responses important
for immune surveillance, as well as toler-
ance.2 Their unique ability to traverse epi-
thelial tight junctions in the intestine3 to
sample luminal bacteria4 implicates DCs
as the immunological rheostat in the gut
mucosa. Indeed, DCs play a critical role
in the induction of colitis,5,6 but they also
participate in mucosal tolerance.7 Besides
the tolerogenic properties of mucosal-
conditioning factors [e.g., transforming
growth factor-β (TGFβ), thymic stromal
lymphopoietin], intestinal CD103 + DCs
subsets have been shown to be migratory8
and able to induce Foxp3 + regulatory T
cells (Tregs).9 It is not known whether
specific bacteria can trigger a tolerogenic
response in mucosal DCs.
Although the potential importance of
DCs in the host response to Helicobacter
pylori was recognized in 1995,10 the
role of gastric DCs in the pathophysiol-
ogy of H. pylori infection has remained
elusive. Studies examining the interac-
tion between H. pylori and DCs have
shown that dendritic cell-specific inter-
cellular adhesion molecule-3-grabbing
 Figure 1. H. pylori stimulates bone marrow-derived DCs and induces higher TLR-2 expression than E. coli- or A. lwoffii-stimulated DCs. PCR array plates
were designed by SuperArray to screen for H. pylori-specific DC tolerogenic mechanisms (34 genes). Day 6 bone marrow-derived DCs35 were pulsed
with 108 live H. pylori SS1, E. coli or A. lwoffii for 18 h (designated HP-DC, EC-DC and AL-DC). PBS-pulsed DCs (PBS-DC) served as the control. (A–C) Levels
of bacteria-pulsed DC mRNA expressions were compared to PBS-DC mRNA expression. (mRNA expressions of significant difference are shown in bold
italics. p < 0.05, n = 3). HP-DC upregulated a number of immunomodulatory genes, indicating that HP-DC undergo active programming after H. pylori
challenge. (B) Fold change of TLR-2 mRNA expressions over PBS-DC (n = 3). HP-DC upregulated TLR-2 expression, whereas EC-DC and AL-DC down-
regulated TLR-2 expression, implicating the differential programming of HP-DC compared to EC-DC and AL-DC.

nonintegrin (DC-SIGN) is the DC sur-
face receptor that binds H. pylori gly-
cans11,12 and that H. pylori stimulates
DCs predominantly through a Toll-like
receptor (TLR)-dependent signaling cas-
cade.13 H. pylori-pulsed DCs (HP-DC)
promote a type 1 helper T cell (Th1)
response14-20 and, as shown more recently,
an interleukin (IL)-17-producing helper
T cell (Th17) response.21 Several studies
have suggested the immunoregulatory
effects of H. pylori on DCs. We showed
that H. pylori-secreted factors inhibit DC
IL-12 production.22 Obonyo et al. also
demonstrated decreased IL-12 produc-
tion by DCs cultured with increased H.
pylori inoculums.23 Gringhuis et al. dis-
covered that H. pylori Lewis-Y binding
of DC-SIGN enhanced IL-10 production
by DCs but decreased IL-12 and IL-6
production.24 Emerging clinical evidence
of an enhanced regulatory T cell (Treg)
response in the H. pylori-infected stom-
ach that correlates with mucosal tolerance
in children implicates the critical role of
mucosal Tregs in disease modulation.25
In our article published in Gastro-
enterology,26 we provided evidence to sup-
port the hypothesis that an interaction
between H. pylori and mucosal DCs leads
to the higher Treg response observed in
H. pylori-infected patients.25,27 We first
demonstrated that DCs are found in the
normal gastric mucosa using an advanced
confocal microscopy technique called
two-photon microscopy. This imaging
technology allows enhanced resolution
with deeper tissue penetration. Post-
H. pylori infection, we measured an
increased density of DCs, and we observed
some DCs with projections extending
across the epithelial layer into the gastric
lumen. These observations encouraged us
to focus our attention on the role of DCs
in mediating the host immune response
during H. pylori infection. Gastric DCs
isolated from the lamina propria of H.
pylori-infected animals showed increased

326 Gut Microbes Volume 1 Issue 5

expression of CD103. Subsets of DCs
expressing CD103 have been shown to
exhibit tolerogenic properties.9
Because H. pylori-infected patients were
shown to express higher levels of Foxp3,27
a Treg marker, we hypothesized that an
interaction between H. pylori and DC
would result in a response skewed toward
Tregs. The yield of gastric DCs isolated
from mouse stomach was low, so we used
bone marrow-derived DCs to characterize
further the DC response to live H. pylori,
focusing on the Th17:Treg balance. We
found that H. pylori induced Treg skewing
by DCs and inhibited priming of Th17
responses, resulting in a significantly
lower Th17:Treg ratio compared to that
induced by other gram-negative bacteria
(e.g., Escherichia coli or Acinetobacter lowf-
fii). The ability of HP-DC to induce Treg
skewing is partially dependent on TGFβ
and IL-10. Adoptive transfer of HP-DC
into naïve mice, followed by H. pylori
challenge, demonstrated Treg skewing in Figure 2. H. pylori-stimulated bone marrow-derived DCs induced a higher percentage of Tregs
vivo. The functional significance of Treg than E. coli- or A. lwoffii-stimulated DCs. MACS microbead-sorted naïve CD4+CD25- T cells were co-
skewing by H. pylori was explored further cultured with PBS-DC, HP-DC, EC-DC or AL-DC for 72 h. Percentages of Tregs were determined by
FACS (gated on CD4+ T cells). Results shown represent one of two independent experiments with
by depleting Tregs using anti-CD25 anti-
similar findings. HP-DCs are significantly better than uninfected DCs at converting naïve T cells to
bodies. We found that mice adoptively Tregs, indicating HP-DC have undergone active tolerogenic programming rather than failing to
transferred with HP-DC, followed by H. activate.
pylori challenge, exhibited enhanced H.
pylori-specific Th1 and Th17 responses
that correlated with a significant reduc- H. pylori, thus keeping DCs in an imma- predominantly antiinflammatory.28 Netea
tion in H. pylori colonization. Thus, we ture state. To address this question, we et al. demonstrated the role of TLR-2
concluded that the interaction between H. designed a polymerase chain reaction in Treg skewing in a mouse model of
pylori and DCs results in Treg skewing, (PCR)-array plate to examine DC pheno- Candida albicans infection.29 It has also
lowering the Th1/Th17:Treg ratio, thereby typic markers. We observed that HP-DC been shown that HP-DC express lower
favoring chronic colonization by H. pylori significantly upregulated various costimu- CD86 levels and higher TGFβ levels com-
(Fig. 3). One limitation of this study was latory molecules, cytokines and chemo- pared to levels expressed by EC-DC and
the use of bone marrow-derived DCs to kines, indicating that HP-DC took up H. AL-DC, an expression pattern shared by
characterize the host response to H. pylori. pylori and underwent activation program- tumor-induced tolerogenic DCs.30
Ongoing studies will assess the response of ming (Fig. 1 A-C). This is consistent with
primed DCs to H. pylori in vivo. our previous finding that green fluorescent H. pylori-Pulsed DCs Prime
protein (GFP)-expressing H. pylori were Inducible Treg in Vitro
H. pylori Stimulates Bone taken up by DCs in culture.22 To deter-
Marrow-Derived Dendritic Cells mine the pathways that are specific for Because the peripheral conversion of Tregs
HP-DC to induce Tregs, we compared the occurs in an antigen-specific manner, we
In our study,26 HP-DC were shown to gene expression patterns of EC-DC and speculated that HP-DC, but not PBS-DC,
induce a significantly higher percentage AL-DC. We measured significant upregu- induce Treg conversion. To investigate
of CD25 +Foxp3 + Tregs in vitro compared lation of TLR-2 mRNA in HP-DC, but this further, we sorted naïve CD25- T
to that induced by EC-DC or AL-DC. downregulation in EC-DC and AL-DC cells from mouse spleen and compared the
However, this induction by HP-DC was (Fig. 1D). Thus, we speculate that TLR-2- peripheral Treg conversion by HP-DC to
not significantly different compared to the mediated H. pylori sensing may be impor- that by PBS-DC, EC-DC or AL-DC. As
induction by phosphate-buffered saline- tant in H. pylori-directed DC tolerogenic shown in Figure 2, HP-DC are superior in
treated DCs (PBS-DC), prompting us to programming. This is consistent with priming inducible Tregs, which provides
ask if the ability of HP-DC to induce Tregs microarray findings reported by Rad et further evidence of active programming in
is a result of a failure of DC activation by al., which suggested that DC TLR-2 is HP-DC. Note that EC-DC and AL-DC

www.landesbioscience.com Gut Microbes 327

 bowel disease of 0.64 (95% confidence
interval: 0.54–0.75).34 Proponents of the
hygiene hypothesis attribute this protec-
tive effect to poor environmental hygiene
of developing countries where H. pylori
is endemic. Because H. pylori skews the
immune response towards Treg differen-
tiation, this reduces the susceptibility of
H. pylori-infected individuals to extra-
gastric chronic inflammatory disorders.
Preliminary results in our laboratory indi-
cate that H. pylori influences the mucosal
immune response in the distal bowel and
decreases colitis disease activity. Further
work is underway to elucidate the mecha-
nism involved.

Conclusion

In summary, our work indicates that

H. pylori directs active tolerogenic
programming of DCs and alters the
Th17:Treg balance, favoring chronic
bacterial colonization. Understanding
the mechanism involved in this tolero-
genic programming will shed light on
Figure 3. A model of mucosal immune regulation during H. pylori colonization. Resident CD11c+
mucosal DCs sample luminal H. pylori, upregulate CD103+ and subsequently migrate to stomach- the maintenance of immune homeostasis
draining lymph nodes. H. pylori then instructs DCs to undergo tolerogenic programming, which of gastric mucosal surfaces. Our find-
skews naïve T cells towards increased Treg conversion. This results in a shift in the Th1/Th17:Treg ings also provide a possible explanation
balance, favoring H. pylori persistence. of the negative association between H.
pylori infection and chronic extragas-
are inefficient in priming inducible Tregs. and the functional significance of H. tric or systemic inflammatory disorders
These data suggest that H. pylori directs pylori tolerogenic programming of DCs. (e.g., asthma, lupus and inflammatory
active tolerogenic programming of DCs, This information will lead to a more bowel disease), as the systemic tolero-
rendering them capable of inducing comprehensive understanding of the role genic effect of H. pylori may alter host
H. pylori-specific Treg conversion in the of DCs in mediating mucosal tolerance susceptibility to these disorders. These
periphery. to a specific luminal microbe and set the results may also enhance our under-
stage for the exploration of a dysregu- standing of how different outcomes of
Potential Applications lated microbe-host interaction in disease H. pylori infection in humans affect can-
pathogenesis. cer risk i.e., a tolerogenic responder may
An understanding of how mucosal toler- The demonstration of the Treg skew- develop nonatrophic gastritis and duode-
ance is maintained in the gastrointestinal ing potential of H. pylori also provides a nal ulcers with no elevation of cancer risk;
tract is critical to gain insight into the mechanistic explanation for the numerous whereas a non-tolerogenic responder may
pathogenesis of gut chronic inflamma- epidemiological reports suggesting that develop atrophic gastritis with an elevated
tion. Gut microbiota have been impli- H. pylori infection prevents chronic cancer risk. Further studies will lead to
cated in the maintenance of mucosal inflammatory disorders including a better understanding of how different
immune homeostasis; 31 however, little is asthma 32 and lupus.33 Our own meta-anal- host immune responses alter the suscep-
known about how gut microbes direct ysis and systematic review of 23 studies tibility to cancer.
the induction of tolerance in the gastro- and 5,903 patients also showed a nega-
intestinal tract. We have shown that H. tive association between H. pylori infec- Acknowledgements
pylori actively programs DCs and induces tion and inflammatory bowel disease, This study was partially supported by
peripheral Treg conversion. Future studies with an estimated relative risk of H. pylori a grant from the National Institutes of
will examine the signaling mechanisms infection in patients with inflammatory Health (1 KO8 DK0669907-01).

328 Gut Microbes Volume 1 Issue 5


References
1. Steinman RM, Cohn ZA. Identification of a novel
cell type in peripheral lymphoid organs of mice. I.
Morphology, quantitation, tissue distribution. J Exp
Med 1973; 137:1142-62.
2. Steinman RM. The dendritic cell system and its
role in immunogenicity. Annu Rev Immunol 1991;
9:271-96.
3. Rescigno M, Urbano M, Valzasina B, Francolini M,
Rotta G, Bonasio R, et al. Dendritic cells express
tight junction proteins and penetrate gut epithelial
monolayers to sample bacteria. Nat Immunol 2001;
2:361-7.
4. Macpherson AJ, Uhr T. Induction of protective IgA
by intestinal dendritic cells carrying commensal bac-
teria. Science 2004; 303:1662-5.
5. Berndt BE, Zhang M, Chen GH, Huffnagle GB, Kao
JY. The role of dendritic cells in the development
of acute dextran sulfate sodium colitis. J Immunol
2007; 179:6255-62.
6. Garrett WS, Lord GM, Punit S, Lugo-Villarino G,
Mazmanian SK, Ito S, et al. Communicable ulcer-
ative colitis induced by T-bet deficiency in the innate
immune system. Cell 2007; 131:33-45.
7. Obonyo M, Sabet M, Cole SP, Ebmeyer J, Uematsu S,
Akira S, et al. Deficiencies of myeloid differentiation
factor 88, Toll-like receptor 2 (TLR2) or TLR4 pro-
duce specific defects in macrophage cytokine secre-
tion induced by Helicobacter pylori. Infect Immun
2007; 75:2408-14.
8. Schulz O, Jaensson E, Persson EK, Liu X, Worbs
T, Agace WW, et al. Intestinal CD103 +, but not
CX3CR1+, antigen sampling cells migrate in lymph
and serve classical dendritic cell functions. J Exp Med
2009; 206:3101-14.
9. Coombes JL, Siddiqui KR, Arancibia-Carcamo CV,
Hall J, Sun CM, Belkaid Y, et al. A functionally spe-
cialized population of mucosal CD103 + DCs induces
Foxp3 + regulatory T cells via a TGFbeta and reti-
noic acid-dependent mechanism. J Exp Med 2007;
204:1757-64.
10. Dixon MF. Histological responses to Helicobacter
pylori infection: gastritis, atrophy and preneoplasia.
Baillieres Clin Gastroenterol 1995; 9:467-86.
11. Appelmelk BJ, van Die I, van Vliet SJ, Vandenbroucke-
Grauls CM, Geijtenbeek TB, van Kooyk Y. Cutting
edge: carbohydrate profiling identifies new patho-
gens that interact with dendritic cell-specific ICAM-
3-grabbing nonintegrin on dendritic cells. J Immunol
2003; 170:1635-9.
12. Bergman MP, Engering A, Smits HH, van Vliet SJ,
van Bodegraven AA, Wirth HP, et al. Helicobacter
pylori modulates the T helper cell 1/T helper cell 2
balance through phase-variable interaction between
lipopolysaccharide and DC-SIGN. J Exp Med 2004;
200:979-90.
www.landesbioscience.com Gut Microbes 329
13. Rad R, Brenner L, Krug A, Voland P, Mages J, Lang
R, et al. Toll-like receptor-dependent activation of
antigen-presenting cells affects adaptive immunity to
Helicobacter pylori. Gastroenterology 2007; 133:150-163.
14. Voland P, Zeitner M, Hafsi N, Prinz C. Human
immune response towards recombinant Helicobacter
pylori urease and cellular fractions. Vaccine 2006;
24:3832-9.
15. Guiney DG, Hasegawa P, Cole SP. Helicobacter pylori
preferentially induces interleukin 12 (IL-12) rather
than IL-6 or IL-10 in human dendritic cells. Infect
Immun 2003; 71:4163-6.
16. Hafsi N, Voland P, Schwendy S, Rad R, Reindl W,
Gerhard M, et al. Human dendritic cells respond
to Helicobacter pylori, promoting NK cell and
Th1-effector responses in vitro. J Immunol 2004;
173:1249-57.
17. Kranzer K, Eckhardt A, Aigner M, Knoll G, Deml L,
Speth C, et al. Induction of maturation and cytokine
release of human dendritic cells by Helicobacter pylori.
Infect Immun 2004; 72:4416-23.
18. Amedei A, Cappon A, Codolo G, Cabrelle A, Polenghi
A, Benagiano M, et al. The neutrophil-activating
protein of Helicobacter pylori promotes Th1 immune
responses. J Clin Invest 2006; 116:1092-101.
19. Voland P, Hafsi N, Zeitner M, Laforsch S, Wagner H,
Prinz C. Antigenic properties of HpaA and Omp18,
two outer membrane proteins of Helicobacter pylori.
Infect Immun 2003; 71:3837-43.
20. Rathinavelu S, Kao JY, Zavros Y, Merchant JL.
Helicobacter pylori outer membrane protein 18
(Hp1125) induces dendritic cell maturation and
function. Helicobacter 2005; 10:424-32.
21. Khamri W, Walker MM, Clark P, Atherton JC,
Thursz MR, Bamford KB, et al. Helicobacter pylori
stimulates dendritic cells to induce interleukin-17
expression from CD4 + T lymphocytes. Infect Immun
2010; 78:845-53.
22. Kao JY, Rathinavelu S, Eaton KA, Bai L, Zavros Y,
Takami M, et al. Helicobacter pylori-secreted factors
inhibit dendritic cell IL-12 secretion: a mechanism
of ineffective host defense. Am J Physiol Gastrointest
Liver Physiol 2006; 291:73-81.
23. Obonyo M, Cole SP, Datta SK, Guiney DG.
Evidence for interleukin-1-independent stimulation
of interleukin-12 and downregulation by interleu-
kin-10 in Helicobacter pylori-infected murine den-
dritic cells deficient in the interleukin-1 receptor.
FEMS Immunol Med Microbiol 2006; 47:414-9.
24. Gringhuis SI, den Dunnen J, Litjens M, van der
Vlist M, Geijtenbeek TB. Carbohydrate-specific
signaling through the DC-SIGN signalosome tailors
immunity to Mycobacterium tuberculosis, HIV-1 and
Helicobacter pylori. Nat Immunol 2009; 10:1081-8.
25. Harris PR, Wright SW, Serrano C, Riera F, Duarte
I, Torres J, et al. Helicobacter pylori gastritis in chil-
dren is associated with a regulatory T-cell response.
Gastroenterology 2008; 134:491-9.
26. Kao JY, Zhang M, Miller MJ, Mills JC, Wang B,
Liu M, et al. Helicobacter pylori immune escape is
mediated by dendritic cell-induced Treg skewing and
Th17 suppression in mice. Gastroenterology 2010;
138:1046-54.
27. Rad R, Brenner L, Bauer S, Schwendy S, Layland L,
da Costa CP, et al. CD25 +/Foxp3 + T cells regulate
gastric inflammation and Helicobacter pylori coloni-
zation in vivo. Gastroenterology 2006; 131:525-37.
28. Rad R, Ballhorn W, Voland P, Eisenacher K, Mages
J, Rad L, et al. Extracellular and intracellular pat-
tern recognition receptors cooperate in the recogni-
tion of Helicobacter pylori. Gastroenterology 2009;
136:2247-57.
29. Netea MG, Sutmuller R, Hermann C, Van der Graaf
CA, Van der Meer JW, van Krieken JH, et al. Toll-
like receptor 2 suppresses immunity against Candida
albicans through induction of IL-10 and regulatory T
cells. J Immunol 2004; 172:3712-8.
30. Dumitriu IE, Dunbar DR, Howie SE, Sethi T,
Gregory CD. Human dendritic cells produce TGFbeta
1 under the influence of lung carcinoma cells and
prime the differentiation of CD4 + CD25 +Foxp3 +
regulatory T cells. J Immunol 2009; 182:2795-807.
31. Noverr MC, Huffnagle GB. Does the microbiota
regulate immune responses outside the gut? Trends
Microbiol 2004; 12:562-8.
32. Chen Y, Blaser MJ. Helicobacter pylori colonization is
inversely associated with childhood asthma. J Infect
Dis 2008; 198:553-60.
33. Sawalha AH, Schmid WR, Binder SR, Bacino DK,
Harley JB. Association between systemic lupus ery-
thematosus and Helicobacter pylori seronegativity. J
Rheumatol 2004; 31:1546-50.
34. Luther J, Dave M, Higgins PD, Kao JY. Association
between Helicobacter pylori infection and inflamma-
tory bowel disease: a meta-analysis and systematic
review of the literature. Inflamm Bowel Dis 2010;
16:1077-84.
35. Kao JY, Zhang M, Miller MJ, Mills JC, Wang B,
Liu M, et al. Helicobacter pylori immune escape is
mediated by dendritic cell-induced Treg skewing and
Th17 suppression in mice. Gastroenterology 2010;
138:1046-54.