Helicobacter pylori elicits B7H3 expression on gastric epithelial cells: Implications in local T cell regulation and subset development during infection.

Helicobacter pylori (H. pylori) is a gram negative bacterium that infects more than 50% of humanity and is associated with gastritis, peptic ulcer and gastric cancer. Although CD4+ T cells are recruited to the gastric mucosa, the host is unable to clear the bacteria. Previously, we demonstrated that H. pylori infection upregulates the expression of the T cell co-inhibitory molecule B7-H1 while simultaneously downregulating the expression of T cell co-stimulatory molecule B7-H2 on gastric epithelial cells (GEC), which together affect the Treg and Th17 cell balance and foster bacterial persistence. Because B7-H3, another member of the B7 family of co-inhibitory receptors, has been found to have important immunoregulatory roles and in cancer, in this study we examined the expression of B7-H3 molecules on GEC and how the expression is regulated by H. pylori during infection. Our study showed that both human and murine GEC constitutively express B7-H3 molecules, but their expression levels increased during H. pylori infection. We further demonstrated that H. pylori uses its type 4 secretion system (T4SS) components CagA and cell wall peptidoglycan (PG) fragment to upregulate B7-H3. Th17 cells and Treg cells which are increased during H. pylori infection also had an effect on B7-H3 induction. The underlying cell signaling pathway involves modulation of p38MAPK pathway. Since B7-H3 were shown to up-regulate Th2 responses, the phenotype of T cell subpopulations in mice infected with H. pylori PMSS1 or SS1 strains were characterized. A mixed Th1/Th2 response in H. pylori infected mice was observed. Consistent with previous findings, increased Treg cells and decreased Th17 cells in MLN of PMSS1 infected mice compared to SS1 infected mice was observed. Human biopsy samples collected from gastritis biopsies and gastric tumors showed a strong association between increased B7-H3 and Th2 responses in H. pylori strains associated with gastritis. T cell: GEC co-cultures and anti-B7-H3 blocking Ab confirmed that the induction of Th2 is mediated by B7-H3 and associated exclusively with an H. pylori gastritis strain not cancer or ulcer strains. In conclusion, these studies revealed a novel regulatory mechanism employed by H. pylori to influence the type of T cell response that develops within the infected gastric mucosa.


INTRODUCTION
Helicobacter pylori (H. pylori) colonizes the human gastric mucosa and may induce gastritis, peptic ulcer and two forms of neoplasia: gastric adenocarcinoma and mucosa-associated H. pylori usually causes chronic infection. Though the host mounts an increased CD4 + T cell response but those T cells are hyporesponsive. During H. pylori infection, patients have a mixed Th1/Th2 response, with increased Treg and Th17 cells in their circulation [33][34][35][36][37][38][39]. Though there are reports showing the type of T cell responses elicited by H. pylori infection, there is a gap in our knowledge regarding the mechanism that H. pylori uses to induce different phenotypic subsets of T cells. Previously our group has shown that H. pylori modulates B7 molecule expression in GECs, which not only help restrain T cells responses, but also induce T regulatory (Treg) cells to assist in H. pylori survival [40][41][42]. Our data also showed that H. pylori uses its T4SS to downregulate B7-H2 expression in GEC, which helps to keep Th17 cells in suboptimal levels, since Th17 are important in the control of extracellular bacterial infections, this downregulation of B7-H2 helps H. pylori to persist [41]. We also demonstrated that H. pylori-mediated up-regulation of B7-H1 expression in GEC causes induction of Treg cells, which contributes to the establishment of a chronic infection [42]. In this study, we investigated another important B7 molecule, B7-H3 and showed that H. pylori upregulates the expression of this molecule on GEC. The upregulation of B7-H3 is regulated not only by the T4SS but also by the cytokines produced by Th17 and Treg cells. We further evaluated the underlying cell signaling pathway and demostrate that H. pylori uses the p38 MAPK pathway for B7-H3 upregulation.
H. pylori is one of the most genetically diverse bacterial species. H. pylori strains differ in the rate with which they have cag PAI in their genome. The EPIYA motifs in cagA gene also differ between Asian and western countries. Moreover, H. pylori infection may result in gastritis, ulcer and gastric cancer development. We examined how H. pylori strains isolated from these three type of gastric diseases modulate B7-H3 expression on epithelial cells. In this study we were interested to determine whether the increase of B7-H3 is consistent with all strains or not. Using different H. pylori strains and patient samples from gastritis and tumors we have shown that only H. pylori strains associated with gastritis causes increased B7-H3 expression and induction of the GATA3 + Th2 cell response. This finding was further confirmed by co-culturing GECs infected with different H. pylori strains with naïve CD4 + T cells. This is a novel finding which shows how H. pylori manipulates GECs to differentially express the B7-H3 molecules and thus regulates T cell responses involved in the H. pylori associated immune-pathogenesis to promote bacterial persistence. Future studies will examine how these findings may be applied in vaccine efforts against H. pylori and possibly in prevention or treatment of gastric cancer.

I. Human tissue
Gastric antrum biopsy specimens were obtained from consenting patients undergoing gastroesophageal-duodenoscopy in accordance with an approved Institutional Review Board protocol. GECs were isolated from the biopsy specimens as described previously [41]. Patients were considered infected if H. pylori was detected by rapid urease testing, histopathology, and by culture of H. pylori from biopsies.

II. Cell lines, bacterial cultures and small peptides
Human GECs N87 and AGS were obtained from the American Type Culture Collection (ATCC) and HGC-27 was obtained from RIKEN, The Institute of Physical and Chemical Research, Japan. All cell lines were maintained in RPMI 1640 with 10% fetal bovine serum (FBS) and 2 mM L-glutamine. As representative murine GEC, Immortomouse stomach epithelium (ImSt) cells were maintained in media described by Whitehead et al. [43]. H. pylori strains 51B and 26695 as well as their corresponding isogenic cagA and cag PAI mutants were described previously [41,44]. H. pylori LC-11 and CA8 were originally isolated from the antral mucosa of a patient with duodenal ulcer and gastric cancer, respectively, as previously described [45,46], Tryptic soy agar (TSA) plates supplemented with 5% sheep's blood (Becton Dickinson, San Jose, CA) were used to grow H. pylori strains. Blood agar plates with 2.5 μg/ml of chloramphenicol (Technova, Hollister, CA) were used to maintain cagA - [41] and cag PAIstrains at 37°C under microaerophilic conditions [41]. For the infection of mice H. pylori Sydney strain 1 (SS1) and PM-SS1 (pre-mouse SS1) were used, which were provided by Drs. J. Pappo (Astra) and Richard Peek (Vanderbilt Univ.), respectively [47]. iEDAP (InvivoGen, San Diego, USA), a PG-like molecule that is a NOD1 receptor, was used to investigate the role of PG in B7-H3 expression.

III. Animals
Female six-to-eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were used in the model of gastric H. pylori infection. Animals were tested negative for the intestinal Helicobacter spp. prior to their use in the experiments.

VI. Real-Time RT-PCR
Real-time RT-PCR analysis was performed as previously described [41].

IX. Bio-Plex
The levels of IL-4 from T cell-GEC co-culture were measured using Luminex array (Millipore, Billerica, MA, USA) according the manufactureŕs instruction. Samples were analyzed using Bio-Plex Manager software (Bio-Rad).

X. Statistical analysis
The results were expressed as the mean ± SE of data obtained from at least three independent experiments done with triplicate sets per experiment unless otherwise indicated. Differences between means were evaluated by analysis of variance (ANOVA) using student t test for multiple comparisons and considered significant if p was <0.05.

I. Expression of B7-H3 on gastric biopsies
To determine the expression of B7-H3 in relation to H. pylori infection we isolated GECs from biopsy samples, which were collected from H. pylori infected or from healthy individuals. B7-H3 expression was measured by real time RT-PCR after mRNA was extracted from the samples. Our RT-PCR data showed a strong upregulation of B7-H3 expression in the H. pylori infected biopsies compared to uninfected samples ( Figure 1A).

II. H. pylori T4SS regulate B7-H3 expression on GEC during infection
To evaluate whether B7-H3 upregulation is a direct effect of H. pylori infection and not an indirect result of inflammatory changes in the host, we used GEC lines and infected them with H. pylori. Since H. pylori T4SS has the capacity to modulate GEC homeostasis and because we have seen their effect in the modulation of B7-H1 and B7-H2 molecules [41,49], we used H. pylori 51B wild type (WT) and H. pylori 51B cag PAI mutant strain to infect GEC (N87 cells). B7-H3 expression was measured after 24-hr infection using flow cytometry. A significant upregulation of B7-H3 expression in GEC infected with H. pylori WT but not with H. pylori cag PAI mutant strains was observed, suggesting that H. pylori T4SS plays role in B7-H3 induction ( Figure 1B). To further dissect the role of the effector protein CagA H. pylori 51B cagA mutant was used to infect GEC along with the H. pylori 51B WT strain. Both flow cytometry and RT-PCR data showed that CagA influences B7-H3 upregulation, since in the absence of CagA B7-H3 expression by GECs remained at basal levels ( Figure 1 C, D). These results were also confirmed in different cell lines (AGS, HGC-27) and by using H. pylori 26695 WT and the corresponding isogenic mutant strains (not shown). Furthermore, a murine cell line was used to confirm these findings and to evaluate whether murine GEC express B7-H3 and whether this expression is regulated by H. pylori T4SS or not, before using a murine model. To this end, the murine GEC line (ImSt) were infected with H. pylori PMSS1, which contains a functional T4SS and with H. pylori SS1 strain in which the T4SS is defective and cannot deliver CagA into GEC. Flow cytometry data showed a significant upregulation of B7-H3 expression in murine GEC infected with H. pylori PMSS1 strain but not with the SS1 strain ( Figure 1E). Overall, these data demonstrated a strong correlation between the presence of T4SS, more specifically of the CagA oncoprotein, and induction of B7-H3 expression on GEC.

III. Role of PG in B7-H3 upregulation
Along with CagA, H. pylori T4SS also translocates PG fragments into GECs, which are recognized by NOD1 and cause activation of cell signaling pathways that result in inflammatory mediator release [14][15][16]50]. Further, in H. pylori infection, NOD1 is upregulated and associated with higher inflammation in GC [63]. To determine the involvement of PG in B7-H3 upregulation GECs were treated with iEDAP, which is a PG analogue recognized by NOD1 ligand. B7-H3 expression was significantly upregulated in mRNA level (Figure 2A) after iEDAP stimulation. Flow cytometry was used as an independent approach to measure the upregulated surface expression of B7-H3 ( Figure 2B). Kinetics data showed a progressive upregulation of B7-H3 as early as 18-hr of stimulation which peaked at 24-hr ( Figure 2C), and is mediated by H. pylori T4SS component CagA and PG ( Figure 2D).

IV. H. pylori uses p38MAPK pathway for B7-H3 upregulation
Further analysis was done to determine the cell signaling pathway used by H. pylori for B7-H3 up-regulation. To that end, the cells were treated with different pharmacological inhibitors of NFκB, MAPK, STAT3, PI3K and mTOR pathways. Our data indicated that upregulation of B7-H3 by the H. pylori strain was blocked in the presence of PD169316, which is a p38 MAPK specific inhibitor (Fig. 3). In contrast, inhibition of PI3K, mTOR, STAT3 and NFκB pathways did not affect H. pylori mediated upregulation of B7-H3 expression (Data not shown). These results suggest that p38 MAPK pathway is a key signaling pathway in H. pylori-mediated upregulation of B7-H3 on GECs.

V. B7-H3 expression is regulated by Th17 and Treg cells
Cytokines regulate the expression of immunoregulatory molecules, which allows for fine tuning of the immune response. During H. pylori infection there is induction of Th17 cells [37][38][39]. Since patients have increased circulating levels of IL-17 we sought to investigate the effect of this cytokine on B7-H3 expression. In RT-PCR analysis ( Figure 4A) significant induction of B7-H3 after IL-17 (10 ng/ml) stimulation was observed. Further, the experiments showed that the expression of B7-H3 on the GECs in response to IL-17 stimulation was increased in a dose-dependent manner (1-100 ng/ml) ( Figure 4B). The surface expression of this ligand was also analyzed at different time points (18-hr, 24-hr and 48-hr) after IL-17 treatment. Expression was significantly increased in GECs after 18-hr of incubation with IL-17, which remains constant after 24 h but decreases after 48 h incubation ( Figure 4C).
Treg cells, which are frequently found in H. pylori-infected patients, produce IL-10 and TGF-β [34,35]. Since there is bidirectional regulation of Treg cells and B7-H1 [51], we investigated whether the hallmark cytokines produced by these cells affect B7-H3 expression. To that end we stimulated GEC with either IL-10 or TGF-β alone or in combination. Both IL-10 and TGF-β induced B7-H3 expression on GECs ( Figure 5A and 5B). Flow cytometry data also showed a cumulative effect of IL-10 and TGF-β in B7-H3 expression ( Figure 5C). Taken together, these data suggested that cytokines produced by Th17 and Treg cells play an important role in B7-H3 expression in GEC. Thus H. pylori regulates B7-H3 expression both directly by using CagA cytotoxin and also indirectly by inducing these T cell subtypes.

VI. Different T cell subset development during H. pylori infection
During H. pylori infection there is an increased frequency of Th1/Th17 and T reg cells in the gastric mucosa. Previously we have shown that H. pylori infection upregulates B7-H1 molecule expression by GECs, which in turn helps to induce further Treg cell development [49]. On the other hand, we also showed that H. pylori T4SS mediated downregulation of B7-H2 in GEC, which impairs Th17 cell development [41]. Besides the reported effects of B7-H3 on T cell activation and inactivation, recent studies by Nagashima O et al., showed that B7-H3 can upregulate Th2 responses [26]. Since our data showed H. pylori upregulates B7-H3 expression we sought to investigate whether the modulation of B7-H3 expression affects local T cell responses. To that end, we collected MLN from mice infected with PMSS1 and SS1 strains and analyzed the T cell subsets present by measuring mRNA expression of the different T cell transcription factors considered "master regulators" for each CD4 + T cell subset, such as GATA3, Tbet, RORγ and FoxP3 for Th2, Th1, Th17 and Treg cells, respectively. Mice infected with SS1 strain showed increased induction GATA3, Tbet and RORγ compared to the PMSS1 strain. However, the MLN cells from PMSS1 infected mice showed increased FoxP3 expression compared to those from SS1 infected mice ( Figure 6A). The mRNA expression of the corresponding cytokines produced by Th2, Th1, Th17 and Treg cells, e.g. IL-4, IFN-γ, IL-17A and IL-10, in MLN was further measured. The cytokine data correlate with the transcription factors found in mice infected with the different strains ( Figure 6B).

VII. Increased B7-H3 and GATA3 expression in gastritis patients
A previous report showed the presence of Th2 cells during H. pylori infection [31].
Consistent with that report, herein, the mouse model also showed induction of GATA3 + Th2 cell in MLN after H. pylori infection. Since B7-H3 has been shown to influence Th2 cell development, we sought to determine the influence of B7-H3 induction by GECs during H. pylori infection in Th2 cells response and whether it depends on the infecting strain. To this end, specimens from patients with gastritis and gastric tumors were evaluated. Biopsy samples from gastritis and samples from gastric tumors were evaluated for the relative expression of B7-H3 and GATA3. Interestingly, samples collected from gastritis patients showed increased B7-H3 and GATA3 expression compared to those from healthy individuals. However, in the case of patients with gastric tumors the expression of both B7-H3 and GATA3 was decreased, which suggested B7-H3 and Th2 induction during H. pylori infection might be a characteristic of gastritis strains (Figure 7).

VIII. B7-H3 expressed by GEC after H. pylori infection induces development of Th2 cells
To further confirm whether the induction of B7-H3 and Th2 is only associated with H. pylori gastritis strains, N87 cell lines were treated with either medium alone or with different H. pylori strains: CA8 (from a gastric cancer case), 51B (from a gastritis case) and LC-11 (from an ulcer case [44,45,61]). After 8 h of infection, the cells were washed extensively and incubated with isolated CD4 + naïve T cells for 5 days. T cells were harvested and stained for CD25, Tbet, GATA3, RORγt and FoxP3 monoclonal antibodies and analyzed by flow cytometry. The data showed increased GATA3 + cells in T cells co-cultured with GECs preinfected with the gastritis strain (H. pylori 51B), but not with the other strains ( Figure 8A). A significant increase in GATA3 + Tbet + double-positive cells was also observed in T cells co-cultured with GECs pre-infected with the gastritis strain, suggesting conversion of Th1 cells to Th2 cell type (data not shown). Interestingly, incubation of the T cells with GECs pretreated with blocking B7-H3 antibody reduced Th2 cell frequency. This data suggested that induction of Th2 is influenced by B7-H3 ( Figure 8B).

Discussion
B7-H3 has previously been considered a co-stimulatory molecule which promotes T cell proliferation [17]. But later studies have shown that B7-H3 can also function as a coinhibitory molecule [17][18][19][20][22][23][24][25]. B7-H3 is expressed by an array of cell types. A previous study showed that RSV causes induction of B7-H3 in tracheal, bronchial, and alveolar epithelial cells [21]. We have shown previously that B7-H3 is expressed by GEC [40]. In this study we demonstrate that H. pylori increases the expression of B7-H3 on GEC upon infection. Increased expression of B7-H3 was demonstrated on the GECs isolated from biopsies of H. pylori infected patients. This observation was confirmed in vitro by increased B7-H3 mRNA level and surface expression in a panel of GEC lines (N87, AGS and HGC-27) after infecting with H. pylori 51B and 26695 strains. Although our lab previously showed that GEC express B7-H3, the expression was unchanged after infection with H. pylori LC-11 strain [40]. That observation together with our recent observations suggest that this cellular response to infection might depend on the infecting H. pylori strain since both 51B and 26695 were isolated from patients with gastritis, while LC-11 originated from a patient with peptic ulcer. These observations were further confirmed in this study by using gastric tissue samples from patients with different gastric diseases associated with H. pylori infection. Lina et al. Page 8 H. pylori T4SS is an important virulence factor that influences GEC homeostasis [52,53]. The recent findings by our group regarding the involvement of T4SS by H. pylori to modulate B7 molecule expression [41], led us to consider H. pylori T4SS as a virulence factor responsible for the up-regulation of B7-H3 by GECs. By using H. pylori WT and cag PAI isogenic mutants, we showed here that B7-H3 induction depends on H. pylori T4SS.
This expression pattern was reproduced both in human GECs and murine GEC (ImSt) infected with H. pylori. Besides using a mutant which lacks the whole cag PAI we also used an H. pylori mutant only devoid in the cagA gene to determine the role of this effector protein translocated by the T4SS in B7-H3 induction. Our study showed that induction of B7-H3 depends on the presence of CagA. PG, the other component translocated to GEC by T4SS may act as an inflammatory molecule and induces IL-8 production by GEC [14][15][16].
As these data showed complete dependence of H. pylori cag PAI but partial involvement of CagA on B7-H3 induction, we hypothesized PG, which is also translocated by T4SS, might also influence B7-H3 induction. The addition of PG fragment iEDAP which is recognized by NOD1 showed induction of B7-H3 both at the mRNA and protein levels. Kinetics data showed that B7-H3 is increased within 18-hrs of stimulation by PG fragments. This study also highlighted the involvement of p38 MAPK pathway in B7-H3 induction, which is known to be activated by both PG and CagA [14,54]. Previously it was shown that PG can be modified to resist lysozyme and this mechanism helps H. pylori survival [55]. However, this is the first study showing role of PG in GEC modification and T cell regulation.
Cytokines play an important role in influencing the expression of different immune regulatory molecules. Since IL-17, IL-10 and TGF-β produced by Th17 and Treg cells have been shown to be present in increased amounts in H. pylori infected patient [34,35,[37][38][39], we hypothesized that these cytokines may act in paracrine fashion to affect the induction of B7-H3 on GEC. Our data showed that stimulation of GECs by both Th17 cytokine (IL-17) and Treg cell cytokines (IL-10 and TGF-β) causes increased expression of B7-H3 molecules on GECs. Regulation of other B7 molecules by IL-10 and TGF-β have been shown previously. For instance, IL-10 was shown to inhibit B7 molecule expression in macrophages and B7-2 expression in DCs [56,57]. Also, TGF-β has been found to inhibit B7-1 expression in APCs [57]. Another study showed IL-10 down-regulated B7-1 and B7-2 expression on Mycobacterium tuberculosis-infected monocytes to a greater extent than did TGF-β [59]. However, IL-10 and TGF-β did not show any additive or synergistic inhibition in their study, whereas, in this study, we found TGF-β is a better inducer of B7-H3 than is IL-10, and they have synergistic effects in B7-H3 induction.  [41,49]. Our lab has previously shown that H. pylori-mediated modulation of Th17 and Treg cell responses depends on altered expression of B7-H2 and B7-H1 molecules on GECs [41,49]. Besides being a positive stimulator for T cell activation, B7-H3 has also been shown to play a role in Th2 development and to contribute to pathogenic Th2 cell development during asthma in a mouse model [26]. However, several studies also showed negative regulatory effects of B7-H3 in Th1 and Th2 immune responses [60]. A major question regarding the Th2 cell response observed in H. pylori-infected mice is whether or not this induction of Th2 is influenced by a B7-H3 molecule expression. To answer this question and investigate whether this response depends on the H. pylori strain, samples from H. pylori infected patients with either gastritis or tumor were collected and B7-H3 and GATA3 expression on those samples were compared with samples collected from healthy individuals. Interestingly, the samples collected from gastritis patients, and not from the gastric tumor patients, had increased B7-H3 and GATA3 + cells. Though this study showed a strong association between B7-H3 induction and Th2 development during H. pylori infection, further studies are required to determine the link between disease condition and B7-H3 expression. To further evaluate this finding, a GEC: T cell co-cultures were used, in which the GECs were pre-exposed to H. pylori strains which originated from gastritis, gastric cancer or peptic ulcer in the presence of anti-B7-H3-blocking antibody or control antibody. The flow cytometry data indicated the induction of Th2 cells and Th1/Th2 doublepositive cells in the T cells co-cultured with H. pylori 51B (from a gastritis case) pre-treated cells, suggesting a shift of Th1 towards Th2 cells. Moreover, by using anti-B7-H3 blocking antibody, we showed that induction of Th2 depends on B7-H3.
In conclusion, this study revealed a novel mechanism that H. pylori uses to foster host chronic inflammation in the form of gastritis. This is an important finding which helps to better understand the interaction of H. pylori with GECs and how H. pylori manipulates the host T cell response. The relationship of H. pylori-mediated B7-H3 induction and disease conditions must be further defined.   B7-H3 up-regulation by H. pylori depends on p38 MAPK pathway. B7-H3 expression on GEC was measured by flow cytometry after treating the cells with p38 MAPK inhibitor (PD169316 10 μM/ml) for 1 h and infected with H. pylori for 24 h. The means ± SD are shown as the results of duplicates in four experiments, n=8, * P < 0.05, ** P < 0.01 and *** P < 0.001.  B7-H3 expression is regulated by Treg cell cytokines. (A) Flow cytometry analysis of GEC cells stained for B7-H3 after exposure to 5 ng/mL IL-10 for 24 h showed increased expression in a representative histogram where the solid peak is the isotype control (B) Flow cytometry was done to measure B7-H3 expression on GECs after treating the cells with TGF-β (3 ng/ml) for 24 h. (C) Flow cytometry was done to measure B7-H3 expression on GECs treated with either IL-10 (5 ng/mL) or TGF-β (3 ng/ml) or both IL-10 and TGF-β. The data were expressed as the percentage of positive cells. The means ± SD are shown as the results of duplicates of one of four representative experiments: n=8, * P < 0.05, ** P < 0.01 and *** P < 0.001.  B7-H3 and Th2 induction is associated with gastritis. B7-H3 and GATA3 expression in biopsy and tumor samples isolated from gastritis or gastric tumor patients with a history of H. pylori infection.