The 5 × 106 PBMCs were purified from fresh blood samples using Histopaque 1077, and total RNA was extracted using an RNeasy minikit (QIAGEN, Crawley, UK), as per the manufacturer's instructions. cDNA was synthesized using SuperScript II reverse transcriptase with an oligo(dT) primer (Invitrogen). Real-time PCR was performed on a Rotor-Gene 3000 (Corbett Research, Cambridge, UK) using primers described in **Table 1**, with a DyNAmo HS SYBR green qPCR kit (New England Biolabs, Ipswich, UK) as previously described (48). Amplification was over 45 cycles of 15 s at 95°C, 30 s at 62°C, and 30 s at 72°C. No template controls were included in each run, and a melting curve analysis was performed. First-stage RT-qPCR samples, produced without reverse transcriptase, were assayed in parallel. Results were analyzed using the Pfaffl method (74). *FOXP3* and *IL10* expression levels were normalized against *GAPDH*, comparing to a pooled reference sample from five *Hp*-negative donors to obtain a fold difference. A commercial pooled human cDNA standard (BD Biosciences) was included as a positive control in all assays.

### Human IgE Assays

Plasma IgE concentrations were determined using a Human IgE ELISA Quantification Kit (Bethyl, USA) as per the manufacturer's instructions. Optical densities were measured at 450 nm with a reference wavelength of 595 nm. Concentrations of IgE were determined from a standard curve on each plate. Allergenspecific IgE assays [timothy grass pollen, birch tree pollen, and *Dermatophagoides pteronyssinus* (house dust mite)] were carried out by the Department of Immunology, Queen's Medical Centre, Nottingham, using the ImmunoCAP system (Phadia AB, Uppsala, Sweden).

*Sequences are shown in the 5*′*–3*′ *orientation.*

## *In Vitro* PBMC Culture and Manipulation of the Cytokine Response

A method to induce IgE synthesis in PBMC cultures was adapted from previously published studies (75–77). PBMCs from 5 *H. pylori*-positive and 10 *H. pylori*-negative healthy non-allergic donors were resuspended in RPMI 1640 medium (supplemented with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml transferrin, and 4 μg/ml insulin). The 200-μl aliquots containing 3 × 105 cells were added to the wells of a sterile 96-well polystyrene round-bottomed plate (Thermo Scientific). In order to induce Th2-skewed conditions, rIL-4 and sCD40L were added to the cells at final concentrations of 50 μg/ ml and 100 ng/ml, respectively (Peprotech). The PBMCs were cultured for 12 days at 37°C and 5% CO2. Supernatants were then removed and stored at −80°C.

In order to observe the effects of IL-10 and TGFβ on IgE synthesis, on day 0 of the culture, 20-μl recombinant human IL-10 (rIL-10) (AbD Serotec) or rTGFβ (AbD Serotec) were added at various concentrations to appropriate wells in quadruplicate (76). The equivalent volume of medium was added to control wells. Cytokines were blocked by adding anti-IL-10 (rat IgG1 clone JES3-9D7, eBioscience) and anti-TGFβ (mouse IgG1 clone 1D11, Abcam) mAbs at varying concentrations. The respective isotype control antibodies were added at the equivalent concentrations. The 20-μg/ml JES3-9D7 has previously been shown to efficiently neutralize endogenously produced IL-10 in a similar culture system (77). The neutralization dose (ND50) of 1D11 antibody is typically 0.25–1.25 μg/ml in the presence of 1 ng/ml recombinant human TGFβ1 (78). The PBMCs were then cultured for 12 days at 37°C with 5% CO2 before collection and assay of the supernatants for IgE.

#### Statistical Analysis

Statistical analyses were carried out using Prism 6.00 (GraphPad Software, CA, USA). Since the data were not normally distributed, non-parametric analysis methods were used. Statistical tests of paired data were carried out using the Wilcoxon signed rank test. For unpaired data, the Mann–Whitney *U*-test was used. A significant difference was taken at *p* ≤ 0.05.

Box and whisker plots depict the medians and interquartile ranges in the boxes, with whiskers extending to the maximum and minimum points in the data.

### RESULTS

## Peripheral Blood Th1 and Treg Responses in Infected and Uninfected Patients

Previous work showed a high-level Treg and Th1 response in the *H. pylori*-infected human gastric mucosa and significantly increased numbers of CD4+CD25hi Tregs in peripheral blood (48, 68). We further investigated the circulating Treg population and determined if higher numbers of Th1 cells were also present. We began by quantifying the cytokines IFNγ, IL-10, and TGFβ1 in PBMC culture supernatants from infected and uninfected patients, and comparing the frequencies of Treg and Th1 cells by flow cytometry. PBMCs from all patients secreted IFNγ when stimulated with *H. pylori* lysate antigen (*Hp*) (**Figure 1A**) or the positive control mitogen conA; however, the concentrations were higher (median difference: 3.6-fold) among the *Hp*-stimulated supernatants from infected compared to uninfected individuals (*p* = 0.05). This is indicative of a systemic Th1 response to *Hp* infection. Similarly, there were slightly higher concentrations of IL-10 (median: 1.3 fold, *p* = 0.02), but there were no differences in TGFβ1 levels.

Using flow cytometry to quantify *Hp*-specific Th1 cells, we immunostained for the early activation marker CD69 (72) and analyzed cells that were activated by *Hp* lysate antigen (**Figure 1B**). Higher frequencies of CD69hiCD4+ events were obtained from the *Hp*-stimulated PBMCs of infected compared to uninfected donors (median: *Hp*+ 10.1%, *Hp*− 4.9%; *p* = 0.001), demonstrating a higher frequency of *Hp*-specific systemic T-cells during infection (data not shown). When cells from both infected and uninfected donors were stimulated with *Hp*, there was a significantly increased frequency of CD69hiCD4+IFNγ+ events compared to unstimulated PBMCs (paired analyses: infected *p* = 0.005; uninfected *p* = 0.001; **Figure 1C**), and similar statistically significant trends were found following PMA/ionomycin stimulation. No significant differences in the responses of cells from infected and uninfected donors were found; therefore, we could not confirm an increased frequency of Th1 cells in the peripheral blood of the infected patients. There were also no differences according to the *cagA* virulence genotype of the colonizing bacterial strains (not shown).

To quantify the Treg response, CD25hi staining was taken as a higher intensity of fluorescence compared to that observed for CD4 negative cells (**Figure 2A**), included the FOXP3+ and IL-10+ populations (**Figures 2B,C**), and corresponded with the CD25 staining intensity in the CD127lo population (**Figure 2D**). As expected, not all CD4+CD25hi events expressed FOXP3. 48–81% of gated CD4+CD25hiIL-10+ events were FOXP3+ (**Figure 2E**). A 2.5-fold higher median frequency of CD4+CD25hi cells (*p*= 0.002) was present in PBMCs from infected patients (**Figure 3A**). There was also a 2.5-fold increased level of IL-10+ CD4+CD25hicells (*p* = 0.007; **Figure 3B**). There was a modest 1.7-fold increase in the frequencies of CD4+CD25hiCTLA-4+ cells (*p* = 0.007, data not shown), but there was no difference in the frequencies of CD4+CD25hiGITR+ cells (not shown). The proportion of FOXP3+ events among CD4+CD25hi cells was not different between infected and uninfected donors, or with/without antigen stimulation (**Figure 3C**), and *FOXP3* mRNA levels were no different when assaying PBMCs by RT-qPCR (**Figure 3D**). The variation in FOXP3+ cell frequencies was extremely wide within both the infected and the uninfected groups, which hampered our ability to detect statistically significant differences. The frequencies of IL-10+ and FOXP3+ CD4+CD25hi cells and the corresponding *IL10* and *FOXP3* transcript levels did not vary significantly according to the age or gender of the patient groups; however, those with gastric or duodenal ulcers had significantly lower frequencies of CD4+CD25hi cells than who only had gastritis (medians: 1.80 and 3.89%, respectively; *p* = 0.047). Similar findings were obtained when analyzing CD4+CD25hiIL-10+ data (medians: 0.10 and 0.17%, respectively; *p* = 0.041). In contrast, the proportion of FOXP3+ events among CD4+CD25hi cells was not significantly different with respect to peptic ulcer disease status among the infected group. Stimulating PBMCs from infected patients with

20 μg/ml conA as a positive control stimulus. IFNγ, IL-10, and TGFβ1 concentrations were determined using commercial ELISA kits. Bars depict the median and error bars show the 95% confidence interval. Following conA stimulation, median concentrations from the PBMCs of infected and uninfected patients were, respectively, 1885 and 2035 pg/ml IFNγ, 375 and 393 pg/ml IL-10, and 352 and 306 pg/ml TGFβ1. Th1 cells were quantified in PBMCs from 34 infected and 24 uninfected patients using flow cytometry. (B) Example flow cytometry dot plots of gated CD4+ cells from an infected donor, stained for CD69 and IFNγ after culture of PBMCs in medium alone, or with *Hp* lysate antigen. Quadrant indicates the position of CD69hi IFNγ+ events, with the lower left quadrant containing both CD69-negative and -positive events. (C) The percentage of CD4+CD69hi IFNγ+ events among PBMC lymphocytes is shown following culture in medium alone (unstimulated) or with *Hp* lysate antigen. When stimulated with PMA and ionomycin as a positive control stimulus, the median percentages of CD4+CD69hiIFNγ+ events in PBMCs from infected and uninfected donors were 1.13 and 1.17%, respectively. The percentages of CD4+CD69hiIFNγ+ events were not significantly different between *Hp*-stimulated samples from infected and uninfected donors.

*Hp* antigen resulted in a significant increase in the proportion of Tregs secreting IL-10 (*p*= 0.01), but there was no significant effect on cells from uninfected patients (**Figure 3E**). This indicates that *H. pylori* infection induces a specific IL-10-secreting peripheral blood Treg response. *IL10* mRNA levels in freshly isolated PBMCs from infected patients were 3.8-fold higher than the uninfected group (*p* = 0.001; **Figure 3F**), and there was a 4.8-fold higher mRNA level in the samples from infected patients without peptic ulcer disease compared to those who had ulcers (*p* = 0.017). These data confirm the presence of increased numbers of IL-10+ cells in the circulation of *H. pylori*-infected patients, particularly when peptic ulceration is absent. Since there was an elevated systemic IL-10-secreting Treg response in infected patients, we hypothesized that as in the mouse, this could be an important mechanism behind the protective associations with allergy.

## Association between the Human Regulatory T-Cell and IgE Responses

Total and allergen-specific IgE concentrations in the plasma of 49 infected and 46 uninfected patients were not significantly different (**Table 2**); however, among the infected group, there were differences in total IgE according to the virulence genotype of the colonizing *H. pylori* strains. This was important to evaluate, since presence of a strain expressing CagA has previously been reported to be associated with greater protection against asthma (9). VacA is known to be protective against allergy in mice (45). Since *cagA*+ strains usually also express the most active i1 form of *vacA* (71), we investigated how IgE levels related to these virulence factors. Apart from in five cases, the 29 *cagA*+ strains were also of the more active *vacA* i1 type. All but 5 of the 20 less pathogenic *cagA*− strains were typed as having the

less active i2 form of *vacA*. We were therefore unable to pick apart the roles of these factors individually. The patients infected with the more virulent *cagA*+ or *vacA* i1 strains had threefold lower concentrations of IgE compared to those with *cagA*− or *vacA* i2 strains (**Figure 4**; *p* = 0.005 and *p* = 0.029, respectively). Surprisingly, however, there were no significant differences in Treg frequencies or IL-10 transcripts between those with *cagA*+ versus *cagA*– strains, and the same result was found for *vacA* genotypes (data not shown).

When comparing the magnitude of the Treg response with IgE levels, we observed some interesting trends. Patients with the highest frequencies of CD4+CD25hi PBMCs had the lowest total IgE concentrations. The effect was stronger among the samples from *H. pylori*-positive patients (**Figures 5A,C**), where having a high-level Treg response was more common. The data were stratified according to whether or not CD4+CD25hi cell frequencies were above or below 10%, which equated to the mean + 1 SD of all samples tested and was well above the normal range for frequencies of these cells in peripheral blood as previously reported in other studies (79, 80). Among the samples from infected patients with high CD4+CD25hi cell frequencies, there was a fivefold lower median IgE concentration, compared to those with frequencies below 10% (*p* = 0.001) (**Figure 5C**). Among the samples from infected patients with high IL-10+ CD4+CD25hi cell frequencies (>0.4%, mean + 1 SD), similarly there was a fivefold lower median IgE concentration, compared to those with low IL-10+ Treg frequencies (*p* = 0.01) (**Figure 5B,D**). There was no significant difference in the total plasma IgE concentrations of uninfected patients with high and low Treg frequencies (**Figures 5C,D**).

A significantly reduced grass (*p* = 0.004) and birch (*p* = 0.002) pollen-specific IgE concentration was present among infected patients with high frequencies of peripheral blood Treg cells (**Table 3**), but there were no significant differences among the uninfected group, or with regard to house dust mite-specific IgE. Similar trends were observed when comparing IgE concentrations with frequencies of IL-10+CD4+CD25hi PBMCs (data not shown). There were no significant trends when PBMC Th1 responses were compared with IgE levels (data not shown). Together, these data indicate that *H. pylori* infection is associated with unusually high circulating Treg levels, which could perhaps directly or indirectly suppress IgE production and influence the development of allergy.

Figure 3 | Regulatory T-cells in the peripheral blood of 49 *H. pylori*-infected and 58 uninfected patients. The percentage of CD4+CD25hi events among gated lymphocytes (A). For determination of IL-10+CD4+CD25hi Tregs (B), PBMCs were cultured in the presence and absence of *Hp* lysate antigen. Median frequencies of IL-10+CD4+CD25hi events among unstimulated cells were 0.04% (uninfected donors) and 0.04% (infected donors). The proportion of CD4+CD25hi cells expressing FOXP3 (C) and IL-10 (E) was assessed in PBMCs cultured with and without *Hp* lysate antigen (Unstim and Hp-stim, respectively). RT-PCR was performed on freshly isolated PBMCs from 48 infected and 26 uninfected patients to quantify *FOXP3* (D) and *IL10* (F) mRNA expression levels. Data were normalized relative to *GAPDH* expression. NS = no significant difference.

#### strains, 24 were of the i1 *vacA* genotype and 5 were *vacA* i2. Of the 20 *cagA*− strains, 15 were *vacA* i2 and 5 were of the *vacA* i1 genotype. Points represent the results from individuals and horizontal lines depict the median.

## Mechanistic Experiments to Investigate the Role of Th1 and Treg Cytokines in the Suppression of IgE

To test the previously observed associations between *H. pylori* infection, serum IgE levels and abundance of peripheral blood Treg cells, an *in vitro* culture system was employed. PBMCs from 5 infected and 10 uninfected healthy donors (all without ulcers) were cultured under Th2-skewing conditions [with IL-4 and rCD40L (75)] for 12 days. Total IgE, IL-10, and TGFβ1 concentrations in the culture supernatants were then measured by ELISA. These culture conditions were used to investigate the hypothesis that PBMCs from *H. pylori*-positive donors would be inherently less able to respond to the Th2 stimulation compared to those from *H. pylori*-negative donors, possibly due to IL-10 or TGFβ inhibition of IgE production *in vitro.* The culture system provided an opportunity to investigate the consequence of adding or selectively blocking IL-10 and TGFβ. From the *in vivo* data, we hypothesized that blocking IL-10 would increase IgE production more markedly when the PBMCs were from *H. pylori*-positive donors. As anticipated, IgE concentrations were lower for the infected donors compared to those who were not infected (medians 25.8 and 34.2 pg/ml, respectively; *p* = 0.013; **Figure 6A**). The concentrations of IL-10 were fivefold higher in the supernatants of PBMCs from the infected donors (*p* = 0.055; **Figure 6B**), but there were no differences in the concentrations of TGFβ1 (**Figure 6C**).

The cultures were also performed in the presence or absence of varying concentrations of recombinant IL-10, TGFβ1, or antibodies known to block the function of these cytokines. A range of concentrations was used so that we could investigate if there was a dose–response effect. Comparing each treatment to the non-treated control for each donor, the percentage decrease in total IgE resulting from the addition of recombinant cytokines was calculated. Adding the recombinant cytokines, at a range of concentrations reported previously to potently interfere with human T-helper cell activity (76, 81), caused a dose-dependent reduction in IgE secretion by PBMCs from both *H. pylori*-positive and -negative donors (**Figures 7A,B**). rIL-10 appeared to have a more potent effect than rTGFβ. Adding as little as 10 ng/ml rIL-10 resulted in a median 31% reduction in IgE concentration in the cultures compared to controls without rIL-10 (*p* = 0.020). Adding 20 ng/ml rIL-10 resulted in a median 52% reduction in IgE (*p* = 0.001). In contrast, a significantly reduced IgE concentration could only be achieved using the highest concentration of 100 ng/ml rTGFβ (5% reduction compared to no rTGFβ controls, *p*= 0.041). No differences in IgE response could be found between the PBMCs from *H. pylori*-positive and -negative donors in these assays however.

Adding IL-10 mAb, at a range of concentrations reported previously to block cytokine activity (77, 78), resulted in increased IgE production by PBMCs from both groups (*H. pylori*-positive and -negative donors), compared to cultures with the equivalent concentration of isotype control antibodies (**Figure 7C**). Adding anti-TGFβ had very little effect (**Figure 7D**). The difference in the size of the effect from IL-10 blockade between *H. pylori*positive and -negative donors was statistically significant (20 μg/ ml: 24%, *p* = 0.02; 40 μg/ml: 53%, *p* = 0.006). Therefore, we had confirmation that blocking IL-10, which was present at higher concentrations in the isotype control cultures of PBMCs from *H. pylori*-positive donors (**Figure 6B**), had a pronounced suppressive influence on IgE production *in vitro*.

## DISCUSSION

This study aimed to further investigate the inverse association between *H. pylori* infection and allergy in humans, and fill an important gap in current literature by exploring the potential mechanisms behind this. We were able to demonstrate a link between IL-10 secreting peripheral blood Treg cell numbers and the concentration of IgE in the plasma of infected patients, and we also showed that IL-10 produced by leukocytes from those with *H. pylori* played an important role in controlling IgE production *in vitro*.

Our previous work found an increased Treg and Th1 response in the *H. pylori*-infected human gastric mucosa (48) and higher frequencies of Tregs in the peripheral blood of infected patients (68). These cell types have previously been implicated in the suppression of Th2 responses and allergy. We hypothesized that increased levels of Treg cells and/or Th1 cells in the circulation

CD4+CD25hi Tregs (B) in PBMCs from 49 infected and 46 uninfected donors was assessed by flow cytometry after 15 h culture with *Hp* lysate antigen. This was compared to the total plasma IgE concentration for each patient. The data were divided according to whether there were high (>10%) or low frequencies of CD4+CD25hi lymphocytes (C) and high (>0.4%) or low frequencies of IL-10+CD4+CD25hi events (D) to further examine associations with total IgE concentrations. NS = no significant difference.

Table 3 | Allergen-specific IgE levels in the plasma of 49 infected and 46 uninfected patients with high and lower peripheral blood CD4**+**CD25hi cell frequencies.

*Allergen-specific IgE data were stratified according to whether the patient had a high Treg response (*>*10% CD4*+*CD25hi PBMCs) or if the frequency was below this level. A Mann–Whitney U-test analysis was performed to examine differences in the IgE responses of these groups. NS* = *no significant difference.*

influence immune responses at extra-gastric sites in the body and may play a role in preventing an elevated IgE response. Initially, we compared the IFNγ, IL-10, and TGFβ1 responses of PBMCs to stimulation with *Hp* lysate antigen. Enhanced concentrations of IFNγ [as shown previously (82)] and IL-10 were found in the PBMC supernatants from infected patients, indicating that peripheral blood Th1 and Treg responses were increased. Cells from both *H. pylori*-positive and -negative patients secreted IFNγ and IL-10 in response to *Hp* lysate antigens, indicating that the response was not antigen specific. Flow cytometry was therefore used to determine whether increased frequencies of Th1 and Treg cells were present in the blood of infected patients.

Our experiments employed detection of CD69 expression, an early activation marker (83), as a tool to identify IFNγ+CD4+ Th1 cells that were responsive to *Hp* stimulation. This could be a consequence of antigen-specific reactivation of memory cells or T-cell receptor-independent immune activation. In agreement with the work of Lundgren et al. (84), peripheral blood CD4+ cells secreted IFNγ when stimulated with *Hp* antigen; however, there were no differences between the samples from infected and uninfected donors. The response is therefore unlikely to be due to reactivation of *H. pylori*-specific memory cells, but could arise because of shared or cross-reactive antigens with other bacteria, or because ligands for pattern recognition receptors were present in the antigen preparation (85).

In agreement with our previous work, and that of others, we found higher levels of CD4+CD25hi lymphocytes among PBMCs from the infected donors (68, 84). It was necessary to determine if these populations could be classed as Tregs by using a selection of cellular markers, and a large proportion of the CD4+CD25hi events expressed FOXP3 and/or IL-10. Since enhanced FOXP3+ Treg levels are present in the infected human gastric mucosa (48, 49, 51), we were surprised to find no difference in the frequencies

of FOXP3+CD4+CD25hi PBMCs, but this result was confirmed using RT-qPCR. FOXP3 is not a completely reliable marker for human Tregs, since its expression can be induced in activated cells that lack suppressive function, and not all Treg populations are FOXP3+ (56, 86). Interestingly, we were able to show increased levels of CD4+CD25hiIL-10+ lymphocytes in the PBMCs of infected patients. When stimulating PBMCs with *Hp* lysate antigen, a higher proportion of the Tregs from *H. pylori*-positive patients expressed IL-10. There was no effect on the CD4+CD25hi population from uninfected patients, indicating the presence of a *H. pylori*-specific circulating Treg response. We were also able to confirm this by RT-qPCR, where higher levels of *IL10* mRNA were found in PBMCs from the infected patients. This agrees with several previous reports of *H. pylori*-specific IL-10-secreting CD4+

cells in the blood (87–89). We previously found increased levels of IL-10+FOXP3+ Tregs in the *H. pylori*-infected human gastric mucosa, cellular FOXP3 expression was of high level and could be confirmed by RT-qPCR (48). Our data suggest that slightly different Treg populations may be present in the blood, and these could include a higher proportion of FOXP3− subtypes, such as Tr1 cells (which are IL-10+) (90) and FOXA1+ cells (associated with IFNβ responses) (91), since *H. pylori* stimulates the production of IFNβ by gastric epithelial cells (92). Recently, we were able to show that significantly higher proportions of peripheral blood Tregs from infected patients expressed the chemokine receptor CCR6 and that high concentrations of its ligand, CCL20, were present in infected human gastric mucosal tissue. The CCL20–CCR6 axis was shown to play an important role in the migration of Tregs to the infected gastric mucosa (68). Some studies have suggested that CCL20/CCR6 interactions are important in the development of Th2 responses and allergy (93, 94). It may therefore be the case that CCR6+ Tregs in the circulation of those infected with *H. pylori* are more capable of migration to the site of allergic responses.

Increased concentrations of IL-10 (but not IFNγ) are present in the serum of *H. pylori*-infected patients (95), and we have found high levels of *Hp*-specific IL-10-secreting Tregs in the circulation. This makes it a likely possibility that such cells could cause bystander suppression of other unrelated extra-gastric immune responses *via* IL-10 secretion. Since the major function of Tregs is to prevent autoimmune disease caused by an overzealous immune response (58), we hypothesized that the *H. pylori*-induced Treg response is involved in protection against allergy. Increased IL-10 responses have been postulated as a mechanism behind *H. pylori*mediated protection from allergy in humans (96); however, Cam et al. found that PBMC IL-10 production was not influenced by *H. pylori* status or presence of atopy in children (97). Because of these controversies, we examined the relationship between plasma IgE concentrations and the frequency of IL-10+ CD4+CD25hi Tregs among PBMCs in infected and uninfected individuals. No significant differences in IgE concentrations (total or antigen specific) were found between the groups of infected and uninfected donors. This may be due to collecting samples from patients with GI symptoms, where only a small proportion had concentrations of serum IgE above the normal range, and none had reported that they suffered from allergies or asthma. When associations between Treg frequency and plasma IgE concentration were examined, as anticipated from the literature (76, 98), higher Treg responses coincided with low IgE levels. This trend was exaggerated among the *H. pylori*-positive group, which contained more individuals with high Treg frequencies accompanied by lower concentrations of total, grass pollen-, and birch pollen-specific IgE. The trend was not statistically significant among the uninfected group, indicating that stronger Treg responses in *H. pylori* infection may suppress IgE levels. We acknowledge that as the samples were collected throughout the year, the data are likely to be confounded by seasonal variation in exposure to pollens. Regardless of this, we were able to detect significant differences in the relationship between IgE and circulating Tregs between the infected and uninfected groups.

Blaser et al. reported stronger protective associations between childhood asthma and more virulent CagA+ *H. pylori* infections (7, 9). Our data supported this finding, since those infected with

ELISA. NS = no significant difference.

and horizontal lines depict the median.

*cagA*+ strains had threefold lower plasma IgE concentrations. A number of *H. pylori* virulence factors have been reported to influence the development of allergy in mouse models, including *H. pylori* neutrophil-activating protein (HP-NAP) (a potent stimulator of Th1 responses) and VacA (known to interfere with antigen presentation and to inhibit T-cell activation; also proposed to stimulate Tregs) (45, 99, 100). Strains which express the active i1 form of VacA are usually also *cagA*+ and those expressing the i2 form of VacA are usually *cagA*− (101). This was indeed true in 39/49 of our clinical isolates and, since the *vacA* types also followed the same trend in the IgE data, we consider it likely that the reported stronger protection from CagA+ strains against childhood asthma could actually have been driven by VacA, with CagA acting as a marker for this. We previously found elevated IL-10 responses in the *cagA*+-infected gastric mucosa (48), but in the present study, we could not find a corresponding significant difference in the peripheral blood (data not shown). It has been suggested that *cagA*+ strains may elicit an increased gastric Treg response to modulate the heightened inflammation, but that there is also a pronounced effect on the migration of Tregs, which may have an impact on the populations found in peripheral blood (68, 102).

We performed experiments with an *in vitro* culture system to investigate the impact of IL-10 and TGFβ on IgE production in a well-controlled manner. The healthy donors who provided us with blood samples did not have different plasma IgE concentrations according to their *H. pylori* status (data not shown); therefore, these experiments allowed us to investigate the potential for their PBMCs to be influenced by a Th2-skewing treatment and produce IgE *in vitro.* The cells from the uninfected donors secreted significantly higher IgE concentrations, while producing markedly lower levels of IL-10. This implies that there were marked differences in the PBMC cell populations of infected and uninfected donors and is in agreement with the increased frequencies of IL-10-secreting Tregs among those with *H. pylori.* When rIL-10 was added to the cultures, in accordance with previous data, the PBMCs produced lower amounts of IgE (76). This occurred whether the cells were from infected or uninfected donors. When IL-10 blocking antibody was added, however, there was a significantly increased IgE response, which was especially marked when PBMCs were from infected donors. There were minimal effects from rTGFβ1 or TGFβ antibody blockade. These experiments demonstrate the important role of the *H. pylori*-induced IL-10 response in controlling IgE production.

We acknowledge that a weakness in this study comes from the fact that the blood samples did not come from allergic or atopic patients; however, we have gathered some interesting novel data that confirm and extend previous research on this topic. It is not possible to use an *in vivo* interventional approach in humans to study the role of IL-10 in the protective mechanism behind *H. pylori*-mediated protection from allergy. Our *in vitro* culture system provided some important clues, however, and enables us to move the project forward. Since we wish to understand the effects of *H. pylori* in protection against allergy, the best approach would be to perform an interventional study investigating the effects of antibiotic eradication therapy on allergic parameters and peripheral blood Treg frequencies. This would include looking for differences in IL-10-expressing populations in particular.

In summary, we demonstrated the presence of an increased frequency of regulatory T-cells in the peripheral blood of *H. pylori*-infected individuals. These cells tended to express IL-10, and high-level responses were associated with reduced plasma IgE concentrations. Using an *in vitro* culture system, we showed for the first time that PBMCs from *H. pylori*-infected donors had an enhanced ability to minimize IgE responses, acting in part *via* IL-10 since there was a 50% increase in IgE when IL-10 was blocked. Further *in vitro* work (not shown) found that CD4+CD25hi cells, purified from PBMCs of infected and uninfected donors using magnetic bead separation, were able to suppress proliferation and IFNγ production by CD4+CD25– effector T cells stimulated with anti-CD3/28 beads. This confirms that the cells have a regulatory function, and we are currently working to evaluate whether Tregs from infected individuals have any enhancement in suppressive activity. We also aim to carry out further experiments to elucidate the mechanisms, investigating the types of Tregs responsible (e.g., Tr1 and FOXA1+ cells), and are planning to investigate whether *H. pylori* exerts a long-lasting

#### REFERENCES

influence on the Treg and IL-10 response from early life, or if the immunological effects require continual life-long presence of the infection in the gastric mucosa. Understanding these concepts is going to be of paramount importance, considering the diminishing prevalence of *H. pylori* around the world, and newly reported hopes for an effective vaccine (103, 104).

## AUTHOR CONTRIBUTIONS

KH, DL, AG, RK, JA, and KR designed the experiments; KH, DL, AG, RK, JW, WT, JR, ES, and KR carried out the experimental work; KH, DL, AG, RK, ES, KK, and KR analyzed the data; and KH, DL, KK, and KR wrote the manuscript.

#### ACKNOWLEDGMENTS

The authors thank Mr. S. Martin and Ms. T. Patel for arranging the human IgE tests, and Mrs. N. Lane for technical assistance.

#### FUNDING

Supported by funding from the Medical Research Council [project grant G0601170, Strategic Skills Award G1000401, a Ph.D. studentship for KH, and a Clinical Research Training Fellowship for ES (G0701377)], Nottingham University Hospitals Charities, and the National Institute for Health Research (NIHR), through the NIHR Biomedical Research Unit in Gastrointestinal and Liver Diseases at Nottingham University Hospitals NHS Trust and the University of Nottingham. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the Department of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Hussain, Letley, Greenaway, Kenefeck, Winter, Tomlinson, Rhead, Staples, Kaneko, Atherton and Robinson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Innate Sensing of the Gut Microbiota: Modulation of Inflammatory and Autoimmune Diseases

*Aline Ignacio1 , Camila Ideli Morales1 , Niels Olsen Saraiva Câmara1,2,3 and Rafael Ribeiro Almeida1 \**

*1 Laboratory of Transplantation Immunobiology, Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil, 2Department of Medicine, Nephrology Division, Federal University of São Paulo, São Paulo, Brazil, 3Renal Pathophysiology Laboratory, Department of Clinical Medicine, University of São Paulo, São Paulo, Brazil*

The mammalian gastrointestinal tract harbors a diverse microbial community with which dynamic interactions have been established over millennia of coevolution. Commensal bacteria and their products are sensed by innate receptors expressed in gut epithelia and in gut-associated immune cells, thereby promoting the proper development of mucosal immune system and host homeostasis. Many studies have demonstrated that host– microbiota interactions play a key role during local and systemic immunity. Therefore, this review will focus on how innate sensing of the gut microbiota and their metabolites through inflammasome and toll-like receptors impact the modulation of a distinct set of inflammatory and autoimmune diseases. We believe that a better understanding of the fine-tuning that governs host–microbiota interactions will further improve common prophylactic and therapeutic applications.

*Edited by:*

*Ryo Inoue, Kyoto Prefectural University, Japan*

#### *Reviewed by:*

*Dolores Correa, Instituto Nacional de Pediatria, Mexico Attila Szabo, University of Debrecen, Hungary*

> *\*Correspondence: Rafael Ribeiro Almeida<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

*Received: 03 November 2015 Accepted: 04 February 2016 Published: 19 February 2016*

#### *Citation:*

*Ignacio A, Morales CI, Câmara NOS and Almeida RR (2016) Innate Sensing of the Gut Microbiota: Modulation of Inflammatory and Autoimmune Diseases. Front. Immunol. 7:54. doi: 10.3389/fimmu.2016.00054*

Keywords: microbiota, toll-like receptors, inflammasome, autoimmunity, inflammatory diseases

## INTRODUCTION

The mammalian gastrointestinal (GI) tract harbors more than 500 bacterial species that have protective, metabolic, and trophic roles and are a constant source of stimulation for the immune system (1). Surveillance mechanisms of the innate immune system control the communication of the gut microbiota with the internal environment, preventing their penetration and systemic spread, and maintaining intestinal homeostasis.

The GI innate immune system consists primarily of a physical barrier, which is composed of intestinal epithelial cells (IECs), represented by absorptive enterocytes, mucus-producing goblet cells, hormone-producing enteroendocrine cells, and Paneth cells, which produce antimicrobial peptides and lectins, among other molecules. The selective permeability of the epithelial barrier allows nutrients absorption while contributing to the immune responses by providing microbial products, which can be recognized through pattern recognition receptors (PRRs), including tolllike receptors (TLRs), nucleotide-binding domain leucine-rich repeat-containing receptors (NLRs), RIG-I like receptors (RLRs), C-type lectin family, and AIM2-like receptors (ALRs) (2), triggering different intracellular signaling cascades against pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs).

The relationship between the intestinal epithelium and the gut microbiota is not restricted to prevent host invasion by commensals. Bacteria-mediated fermentation of food that cannot be fully digested in the mammalian intestine releases products such as short-chain fatty acids (SCFAs), which are used as fuel by IECs. In turn, the host provides a stable, nutrient-rich ecosystem for commensals to thrive. The microfold (M) cells are specialized epithelial cells of the mucosa-associated lymphoid tissue found in the follicle-associated epithelium (FAE) that mediate the uptake and transepithelial transport of luminal antigens to intraepithelial immune cells, such as macrophages and dendritic cells, which are responsible for presenting antigens to lymphocytes (3). In the intestinal lamina propria, macrophages and dendritic cell-mediated antigen uptake, either directly from the intestinal lumen by extending projections between IECs or indirectly by M cells, results in cytokine production, which drives either inflammatory Th17 or regulatory T cells, and T-cell-dependent and -independent IgA class-switching responses (4–6).

In this review, we focused on the interactions between the gut microbiota and two distinct groups of molecules, the innate sensors, namely, TLRs and the inflammasomes, which are typically composed of the innate sensors NLRs and ALRs, the adaptor protein apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), and the proinflammatory caspase-1. Increasing efforts have been pointed out toward the understanding of how innate sensing of commensals promotes homeostasis and immunity. Here, we review the impact of innate sensing of the gut microbiota on inflammatory and autoimmune diseases' outcomes.

### TOLL-LIKE RECEPTORS

Toll-like receptors are type I transmembrane proteins with an extracellular horseshoe-shaped domain-containing leucine-rich repeats (LRRs) and a cytoplasmic tail containing a toll/IL-1 receptor (TIR) domain (7). They correspond to one of the five main families of PRRs, which recognize a variety of PAMPs and DAMPs.

Toll-like receptors are capable of forming both homodimers and heterodimers to detect different types of ligands. TLR2/ TLR1 heterodimers recognize triacylated lipopeptides (8), and it has been recently demonstrated that they can also be activated by α-synuclein, a protein associated with Parkinson's disease (9). On the other hand, diacylated lipopeptides are recognized by TLR2/ TLR6 heterodimers (8).

A recent study has shown that human immunodeficiency virus (HIV-1) structural proteins can activate TLR2- and TLR2 related heterodimers (10). A variety of viral proteins from measles virus (MV), hepatitis C virus (HCV), human cytomegalovirus (hCMV), and herpes simplex (HSV) have also been indicated as TLR2 activators (11–14). Lipoteichoic acid (LTA), zymosan, and peptidoglycan also trigger TLR2 signaling when TLR2/ TLR6/CD14, TLR2/Dectin-1, and TLR2/CD14 are assembled, respectively (15–20).

TLR3 recognizes viral double-stranded RNA (dsRNA) (21) and is also capable of recognizing DAMPs released from damaged tissues (22), while TLR7 and TLR8 detect single-stranded RNA (ssRNA) found during viral replication (23–25). TLR4 is the main sensor for lipopolysaccharides (LPS) from Gram-negative bacteria (26) and also detects viral motifs (27). Flagellin is sensed by TLR5 (28), and TLR9 detects unmethylated CpG sequences in DNA molecules of bacterial and viral genomes (29). TLR10 is expressed in humans, but not in mice, making the search for a specific ligand experimentally difficult. It has been recently shown that TLR10 expressed in IECs responds to an unidentified component of *Listeria monocytogenes*, but it requires the presence of TLR2 for inducing NFκB activation (30). This opens the possibility that TLR10 acts as a coreceptor of TLR2, as it is phylogenetically related to TLR1 and TLR6 (31). Furthermore, other authors have demonstrated anti-inflammatory properties of TLR10 expressed in human peripheral blood mononuclear cells (PBMCs) (32).

TLR11 is expressed in mice and as a non-functional pseudogene in humans and has been related to prevention of *Salmonella* infection, the detection of uropathogenic *Escherichia coli*, and the recognition of a profiling-like molecule from *Toxoplasma gondii* (33–35). TLR12 and TLR13 are not expressed in humans but are expressed in mice and are capable of detecting the profiling-like molecule of *T. gondii* and 23S rRNA, respectively (36, 37). Besides PAMPs, endogenous ligands activate TLRs in the absence of infection (38).

Toll-like receptor–ligand interactions lead to dimerization of extracellular domains and results in dimerization of their cytoplasmic TIR domains. This structural modification is recognized by TIR domains on the adaptor proteins myeloid differentiation primary response gene 88 (MyD88), MyD88 adapter-like (MAL)/TIR domain-containing adapter protein (TIRAP), TIR domain-containing adapter-inducing interferon-β (TRIF), and TRIF-related adaptor molecule (TRAM), triggering downstream signaling pathways, which culminate in the expression of inflammatory cytokines, several anti-viral and anti-pathogen proteins, leading to the initiation of the adaptive immune response. The Myd88-dependent signaling pathway is common to all TLRs, except TLR3, which is dependent on TRIF. TLR4 uses both MyD88- and TRIF-dependent signaling pathways (7).

The detection of common molecules shared by pathogenic and non-pathogenic bacteria should trigger the same inflammatory response, but paradoxically, the recognition of commensal bacteria by intestinal receptors is somehow regulated to not result in inflammatory responses but to induce tolerance. Moreover, these TLR-mediated signals contribute to the intestinal homeostasis by regulating IECs proliferation and epithelial integrity (39–42). Interestingly, the localization, distribution, and expression of TLRs in the intestinal epithelium seem to be directly related to their role in maintaining homeostasis (43). IECs have a polarized organization, with an apical pole facing the intestinal lumen and a basolateral one that communicates with other IECs and the lamina propria. TLRs are differentially distributed through the polarized organization of IECs and in different quantities along the entire GI tract.

The human esophagus expresses TLR4, low levels of TLR1 and TLR5, and high levels of TLR2 and TLR3 mRNA in epithelial cells (44, 45). In gastric epithelial cells, it is possible to find expression of TLR2, TLR4, and TLR5, as both mRNA and protein (46). In the human small intestine, it is possible to find mRNA expression from the TLRs 1–10 (47). Under homeostatic conditions, enteroendocrine IECs from intestinal crypts express TLR1, TLR2, and TLR4 (48), and IECs from human colon express relatively high levels of mRNA for TLR3 and less for TLR2 and TLR4 (49).

Colonic IECs are hyporesponsive to LPS, and it may be due to epigenetic mechanisms that downregulate MD-2 and TLR4 expression (50). Moreover, TLR3 in murine IECs seems to have an age-dependent expression related to an enhanced response to rotavirus in adult mice (51). On the other hand, the high basolateral expression of TLR5 is directly induced by administration of flagellin to IECs (52). TLR9 apical expression in colonic IECs has been related to the maintenance of the intestinal homeostasis (53).

The FAE presents a differential expression of TLRs in comparison with villous IECs. The murine small intestine expresses TLR2 and TLR9 in both apical and basolateral sides of the FAE, whereas IECs in the villi shows apical expression only. The TLR5 is abundantly expressed in the apical pole of villous IECs and FAE, whereas TLR4 expression is low (54).

It is conceived that TLR-mediated sensing of microbial products has a dual role in promoting the fine-tune balance between proinflammatory and pro-regulatory immune responses. A better understanding of how these molecules are regulated in face of commensals and pathogenic microorganisms will benefit the design of prophylactic and therapeutic approaches.

## THE DUAL ROLE OF TLRs IN TRIGGERING INFLAMMATORY AND AUTOIMMUNE DISEASES

Bearing in mind the importance of the gut microbiota for the development of the immune system, one might speculate that microbial components could also modulate immunity and trigger inflammatory and autoimmune diseases. Several studies have shown correlations between altered expression of TLRs and inflammatory/autoimmune conditions, and increasing evidence suggests the participation of the gut microbiota during TLRs abnormal signaling and the development of diseases, including type 1 diabetes (T1D), multiple sclerosis (MS), rheumatoid arthritis (RA), and inflammatory bowel diseases (IBDs) (**Table 1**) (55–58).

Rheumatoid arthritis, IBDs, T1D, and MS are classified as multifactorial diseases with genetic and environmental factors contributing to their pathogenesis. Among the environmental factors, the exposition to microbial components has been associated with their development (55, 59–62). The concept called "molecular mimicry" was proposed to explain the role of microbial agents in inflammatory and autoimmune diseases, and according to this concept, cross-reactivity between epitopes from microbes and self-proteins can cause deregulated immune responses, leading to autoantibody production and activation of effectors cells (63).

The major organs exposed to microbial agents and their components are the skin and gut. In the gut, sensing of microbial antigens by innate immune cells and non-immune cells, such as IECs and stromal cells, is mediated by PRRs that recognized PAMPs. It is possible that bacterial components stimulate the mucosal immune cells by penetrating a damaged epithelial barrier or *via*

a paracellular pathway; alternatively, microbial antigens may interact with TLRs at the apical surface of IECs, inducing inflammatory activation of the mucosal immune system. In addition, innate immune cells, such as dendritic cells and macrophages, can also sense PAMPs through TLRs initiating rapid and effective inflammatory responses against microbial invasion (57, 64). As mentioned above, the commensal microbiota is necessary for the constant stimulation of the immune system (1) and TLRmediated sensing of these microorganisms may play a dual role in disease development as a source of both inflammatory and regulatory signals.

Rheumatoid arthritis is an autoimmune disease characterized by chronic inflammation in which cartilage and bone of the affected subjects are progressively destroyed in multiple joints (55). Several immune and resident cells, such as chondrocytes and fibroblasts, contribute to the development and progression of RA. Release of proinflammatory cytokines, such as tumornecrosis factor (TNF)-α and interleukin-1 (IL-1), mainly by macrophages, activation of Th17 lymphocytes, and production of autoantibodies are suggested to play an important role in the disease onset (65). Studies showing the presence of bacterial cell wall components in the joints of RA patients accompanied by changes in their gut microbiota support the idea that commensal bacteria may initiate inflammation in genetically susceptible individuals (66, 67).

Abdollahi-Roodsaz et al. (56) using the IL-1 receptor antagonist-deficient (*IL1rn*−/−) mice that spontaneously develop T-cell-mediated autoimmune arthritis showed that germ-free (GF) *IL1rn*−/− presented no signs of arthritis during 20 weeks of follow-up, whereas matched non-GF animals started to develop the disease from the age of 5 weeks. The authors also demonstrated that the activation of TLRs was dependent on the microbial status of the mice and that TLR2 deficiency in *IL1rn*−/− mice led to less Foxp3+ expression and reduced suppressive activity of Tregs, resulting in an enhancement of clinical and histopathological scores of arthritis. By contrast, *IL1rn*−/−*Tlr4*−/− mice were protected against severe disease. It has been shown that TLR4 contributes to more severe arthritis by modulating IL-17 production and Th17 cell expansion (56, 68). In fact, gut-residing segmented filamentous bacteria (SFB) have been shown to drive both IL-1β and IL-6 production, and Th17 development, which promotes arthritis (69, 70). Therefore, one may conceive that a delicate equilibrium in TLR-mediated sensing of the gut microbiota is necessary to maintain homeostasis and prevent certain autoimmune diseases.

Although the gut microbiota has been extensively explored as an etiologic factor of RA, less is known about oral commensal microorganisms. In this context, the Gram-negative commensal bacterium *Porphyromonas gingivalis* has been a major focus of investigation as it provides a direct link between a specific microorganism and an autoimmune disease (71, 72). In fact, individuals with periodontitis present an increased proportion of *P. gingivalis* and specific antibodies to this bacterium, which has been linked to RA (73). A recent study showed that periodontal disease (PD) induced by *P. gingivalis* increases the severity of experimental arthritis through Th17 induction. This periodontal pathogen induces IL-17-producing T cells through TLR2 activation on

*IL1rn, IL-1 receptor antagonist-deficient mice; MDR1, multidrug resistance gene; UC, ulcerative colitis; IECs, intestinal epithelial cells; CD, Crohn's disease.*

APCs (74), which suggests that the same TLR molecule may play different roles in RA onset, depending on where the molecule is expressed and with which microorganism the interaction occurs. Thus, more studies are still necessary to better determine the role of TLR-mediated sensing of the microbiota and/or pathogens in autoimmune diseases, such as RA. Mice models with organspecific TLR expression could be used to shed light on how TLRs contribute to disease onset depending on their localization.

Inflammatory bowel diseases have been extensively studied in regarding of the gut microbiota–immune system interactions. Two major forms of IBD have been investigated: the ulcerative colitis (UC) and Crohn's disease (CD). Distinctive and complex chronic inflammatory processes characterize both disorders (61, 62), and it has been demonstrated that the intestinal microbiota of patients differ from healthy controls, showing an increase of *Enterococcus* spp. and *Bacteroides* spp. accompanied by a decrease of *Bifidobacterium* spp. and *Lactobacillus* spp. levels (75, 76), suggesting that the gut microbiota may play a pivotal role in intestinal inflammatory diseases.

Increased susceptibility to severe UC has been associated with variants of the multidrug resistance gene (MDR1/ABCB1). Deletion of TLR2 in MDR1A deficiency resulted in fulminant pancolitis, characterized by expansion of CD11b+ myeloid cells, and a shift toward Th1 immune responses in the lamina propria. An unaltered microbiota was required for colitis exacerbation in TLR2/MDR1A double-knockout mice once protection from Ignacio et al. Innate Sensing of the Microbiota

colitis was observed upon antibiotic treatment (77). Treg-specific deletion of MyD88 has been recently shown to culminate in deficiency of Treg cells, increase in Th17 cells, and exacerbated experimental colitis. An overgrowth of SFB and increased bacterial translocation was reported, and also impaired antimicrobial IgA responses (78), suggesting that the TLR-mediated sensing of the gut microbiota contributes to Treg cell-dependent maintenance of the intestinal homeostasis. On the other hand, it has been shown that gut microbiota-mediated triggering of intestinal epithelial TLRs is not critical for promoting intestinal inflammation once mice lacking TNF receptor-associated factor 6 (TRAF6), but not MyD88/TRIF, were protected from dextran sodium sulfate (DSS)-induced colitis (79). Therefore, it is becoming clear that not only the microbiota is important during TLRs signaling through colitis development but also where these molecules are expressed.

Apart from the alterations in the gut microbiota composition, Cario and Podolsky have studied TLRs expression in CD and UC patients and showed variations in the expression of some receptors. During homeostasis, TLR2 and TLR4 are presented in small amounts on IECs and lamina propria cells to minimize microbiota recognition and to maintain tolerance (80, 81). So, signaling through those PRRs may determine the balance between immunity and tolerance. The authors observed that TLR2 and TLR5 are expressed on IECs from non-IBD subjects and remain unchanged in active IBD. Upregulation of TLR2 was observed in inflammatory cells from the lamina propria of active IBD patients; differential expression of TLR3 was verified in UC patients, which presented a basolateral expression on IECs; both UC and CD patients showed an abundant TLR4 expression on basolateral and apical surfaces of IECs, respectively, and enhanced expression of TLR4 was also present in the lamina propria of IBD individuals (80).

In DCs, PRRs stimulation induces IL-23 releases, which is an important component of antimicrobial defense, but when excessively produced it favors proinflammatory T-cell response and reduces Foxp3+ T cell differentiation. In this way, DCs may act as a key player in the initiation, continuation, and control of IBD (82). Conventional DCs (cDC) from UC and CD patients during remission phase showed an increased TLR4 expression. After stimulation, these cells secreted higher amounts of TNF-α and IL-8, and the uptake of LPS started earlier and was higher than in controls (83). These data suggest that an aberrant TLR4 signaling in cDC of IBD patients may result in an inflammatory phenotype during the acute phase.

Paradoxical effects of TLRs activation may be present in IBD. Bacterial DNA induces strong Th1 immune responses with high production of TNF-α and IL-8, which is also found in experimental and human IBD (84). On the other hand, bacterial DNA stimulation of IECs *via* apical TLR9 may result in antiinflammatory effects, with the inhibition of TNF-α and IL-8 secretion, as well as NFκB activation, reducing colitis severity (53). Furthermore, it has been observed in a CD4+ T-celldependent Severe combined immunodeficiency (SCID) transfer model of colitis that pretreatment of donor mice with CpG completely abolished colitis development in SCID recipients in a CpG–TLR9-mediated modulation of T-cell function (85).

Another important protective effect promoted by TLR signaling was recently elucidated by Kawashima et al. (86) showing that dsRNA of lactic acid bacteria (LAB), one major commensal bacteria, triggered interferon-β (IFN-β) production through TLR3 activation pathway and protected mice from experimental colitis. On the other hand, pathogenic bacteria induced much less IFN-β and contained less dsRNA than LAB, indicating that dsRNA was not involved in pathogen-induced IFN-β induction. These results point toward TLR3 as a sensor to commensal bacteria and suggest a mechanism by which this endosomal receptor contributes to anti-inflammatory and protective immune responses.

Paradoxical effects of TLRs participation are also observed in T1D, an organ-specific autoimmune disease in which insulinproducing β cells are mainly destroyed by not only Th1 but also Th17 lymphocytes (87). The autoreactive CD4+ T cells that infiltrate the pancreas present a proinflammatory phenotype, characterized by IFN-γ secretion (88), and support cytotoxic T lymphocytes (Tc), which are responsible for the progressive destruction of β cells (55). Although the later steps of T1D development are well known, the initial steps remain unclear.

The role of TLRs–PAMP interactions during T1D onset has been investigated in several animal models. Studies performed in BioBreeding Diabetes Resistant (BBDR) rats infected with the ssDNA parvoviruses Kilham rat virus (KRV) showed that KRV acts as a TLR9 ligand by upregulating proinflammatory cytokines in pancreatic lymph nodes, thus inducing islet destruction (89). Pretreatment with poly I:C, a TLR3 ligand, acts synergistically with KRV to induce diabetes in 100% of infected rats (90). Also, it has been demonstrated that the activation of APCs through TLR2-mediated sensing of β cells death contributes to T1D initiation in non-obese (NOD) mice (91). In contrast with these findings, Wen et al. (58) investigating the effects of *MyD88* gene disruption on disease incidence and progression in NOD mice showed that MyD88-deficient NOD mice did not develop T1D, and that the observed protection was dependent on commensal microorganisms once GF MyD88-deficient NOD mice developed disease. The authors also demonstrated that the gut microbiota composition was changed by MyD88 deficiency and colonization of GF MyD88-deficient NOD mice with the bacterial community termed "altered Schedler's flora" (ASF), normally present in healthy mouse gut, attenuated T1D. Thus, it can be suggested that in the absence of TLRs signaling, some bacterial groups predominate and induce tolerogenic responses. However, the receptors and signaling pathways involved in microbiota-dependent protection against T1D development remain unclear.

Some protective signals against T1D development triggered by the gut microbiota have been revealed through studies with NOD mice lacking MyD88 crossed with mice deficient for other components of the innate immune response. As ASF bacteria colonization has reduced T1D in GF MyD88-deficient NOD mice, it is possible that several signaling pathways activated by TLR agonists could contribute to protection when MyD88 signaling is absent. In fact, stimulation of TLR3 and TLR4 induces the activation of a MyD88-independent, TRIFdependent signaling pathway (55), which has been pointed out as a negative regulator of immunity. In this context, Burrows and colleagues (92) have demonstrated that TRIF deficiency did not affect T1D development in Myd88 sufficient NOD mice, while double-deficient mice were more susceptible to T1D. Thus, it could be suggested that TLR3- and TLR4-mediated sensing of the gut microbiota participates in the protection against T1D development in MyD88-deficient NOD mice through tolerating mechanisms involving TRIF signaling.

Protective effects from TLR–microbiota interaction have also been demonstrated in MS studies. The MS is an immunemediated chronic disease in which demyelination and axonal damage are caused by infiltration of myelin-specific autoreactive Th1 and Th17 cells into the central nervous system (CNS) in either relapsing/remitting or progressive condition (93, 94). CNS-derived peptides, such as the melanocortin antagonist agouti-related peptide (AgRP) and neuropeptide Y (NPY), are capable of modulating food intake and physiological processes that control nutrient absorption, which may influence the gut microbiota composition. In turn, gut microbiota modulates brain functions by releasing SCFAs and antigens, such as lipopolysaccharide (LPS), polysaccharide A (PSA), and LTA (95).

Miyake and colleagues (96) have investigated whether the gut microbiota was altered in MS by comparing the gut microbiota of 20 patients with relapsing–remitting (RR) MS with that of 40 healthy subjects. They found differences in the relative abundance of both archaea and butyrate-producing bacteria when comparing MS patients and healthy individuals and a significant reduction of clostridial species in MS patients. Interestingly, none of the clostridial species that were significantly reduced in the gut microbiota of MS patients overlapped with other spore-forming clostridial species capable of inducing colonic regulatory T cells, which have been associated with protection from autoimmunity and allergies (97). However, it has been shown that reconstitution of the gut microbiota from antibiotic-treated mice with *Bacteroides fragilis* protected against experimental autoimmune encephalomyelitis (EAE) by a mechanism dependent on PSAinduced IL-10-producing Treg cells, which may rely on TLR2 signaling (98). In fact, *B. fragilis*-derived PSA converts effector CD4+ T cells into IL-10-producing T cells *in vitro* by a TLR2 dependent mechanism (99). On the other hand, TLR4-mediated sensing of LPS has been shown to stimulate *in vitro* differentiation of Th17 cells and subcutaneous LPS injection increased the frequency of IL-17-producing cells in inguinal lymph nodes, worsening EAE (100).

Numerous studies have focused on the role of B cells during MS pathogenesis (101, 102). B cell-deficient C57Bl/6 mice suffer an exacerbate EAE form and transfer of IL-10-producing B cells into IL-10-deficient mice protected them from disease (101). In fact, it has been recently shown that microbiota-driven IL-1β and IL-6 production promotes regulatory B cells differentiation (103). Lampropoulou et al. (102) showed that TLR-activated B cells produce IL-10 in a MyD88-dependent manner by stimulating TLR4 and TLR9 with LPS and CpG oligonucleotides, respectively. These B cells limit the capacity of DC to produce IL-12 and restrain Th1 differentiation. Therefore, new studies are fundamental to elucidate the role of different TLRs in the context of EAE modulation, which seems to depend on microbiota-driven inflammatory and regulatory immune cells.

## INFLAMMASOMES IN THE GUT

Inflammasomes are multimeric protein complexes typically composed of a sensor protein, the adaptor protein ASC, and the proinflammatory caspase-1, which can be triggered by a variety of stimuli associated with infection and cellular stress (104). Inflammasome activation results in recruitment of ASC, proteolytic cleavage, and activation of caspase-1, which leads to process and release of the proinflammatory cytokines IL-1β and IL-18 (105). The majority of inflammasomes contain a NLR sensor molecule, namely NLRP1 (NOD-, LRR-, and pyrin domain-containing 1), NLRP3, NLRP6, NLRP7, NLRP12, or NLRC4 (NOD-, LRR-, and CARD-containing 4). However, other two inflammasomes have been described containing the pyrin and HIN domain-containing protein (PYHIN) family members absent in melanoma 2 (AIM2) and IFN-γ-inducible protein 16 (IFI16) as sensor molecules (106). A non-canonical activation pathway of inflammatory caspases 4/5 in humans and caspase-11 in mice has also been described and depends on intracellular sensing of lipopolysaccharide (LPS) released upon Gram-negative bacteria escape from vacuoles (107).

Inflammasome signaling has been extensively studied in macrophages in different contexts (104, 108), but little is known about inflammasome expression and function in cells located in the gut. Intestinal CD11b+F4/80+ mononuclear phagocytes that normally reside in the lamina propria were shown to be anergic to ligands for TLRs or commensals but to produce IL-1β upon NLRC4 activation after infection with pathogenic *Salmonella* or *Pseudomonas* (109). More recently, *Enterobacteriaceae* and the pathobiont *Proteus mirabilis* were shown to induce robust IL-1β production through NLRP3 activation in newly recruited intestinal Ly6Chigh monocytes upon epithelial injury (110). IECs, the first cellular barrier toward the gut lumen, were also shown to express a variety of inflammasome components such as NAIPs 1, 2, 5, and 6 in mice (hNAIP in humans), NLRC4, NLRP1, NLRP6, AIM2, caspase-1, caspase-11 (-4, in humans), ASC, and IL-18. These inflammasome components contribute to intestinal homeostasis by regulating commensals' ecology, by restricting pathogens, and by restoring epithelial barrier integrity (105). However, further studies are still necessary to shed light on how inflammasomes are regulated in both hematopoietic and non-hematopoietic intestinal cells in face of commensal microorganisms, diet-derived antigens, and pathogens to maintain homeostasis and systemic immunity.

## INFLAMMASOME-MEDIATED SENSING OF THE GUT MICROBIOTA AND INFLAMMATION

The NLRP6 inflammasome has been shown to regulate intestinal microbiota ecology as metagenomic analysis has revealed pronounced dysbiosis in NLRP6-deficient mice (111, 112). Changes in the biogeographical distribution of microbiota are also observed in NLRP6 deficiency, leading to accumulation of commensal microorganisms in the colonic crypts. Reduced IL-18 levels and increased relative abundance of bacterial phyla Bacteroidetes (*Prevotellaceae*) and TM7 have been observed in NLRP6-deficient mice, which present spontaneous intestinal hyperplasia, chemokine (C–C motif) ligand 5 (CCL5)-mediated inflammatory cell recruitment, and exacerbation of DSS-induced colitis. Cross-fostering and cohousing experiments were sufficient to transfer dysbiotic microbiota to neonatal and adult wild-type (WT) mice, leading to increased susceptibility to DSS-induced colitis. Although deficiency in other inflammasome components, including ASC, caspase-1, and IL-18, also led to dysbiosis and exacerbated colitis, deficiency in NLRC4, NLRP10, NLRP12, and AIM2 had no impact on the susceptibility of WT mice to colitis upon cohousing (111). These mice were shown to have a distinct configuration of their microbiota when compared to NLRP6-deficient mice, which might explain the different results and suggests that although inflammasomes share many of their effector molecules, different impacts on microbiota ecology can be observed.

It has been recently shown that mice deficient in NLRP6 and the inflammasome components ASC and caspase-1 lack a thick continuous overlaying inner mucus layer in the gut due to abrogated mucus secretion by IECs and are unable to clear enteric pathogens from mucosal surface (113). As mucus has an important role in regulating host–microbial interactions, it is likely that dysbiosis and increased intestinal inflammation observed in NLRP6 mice are consequences of this reduced mucus secretion by epithelial cells. Deficiency in NLRP6 and IL-18 has also been linked to colitis-related colorectal cancer (CRC) development. Enhanced tumorigenesis in these mice was dependent on microbiota-induced CCL5-driven inflammation and local IL-6 production and could be transferred to WT mice upon cohousing (114).

Corticotropin-releasing hormone (CRH)-mediated reduction of intestinal NLRP6 expression in mice exposed to wateravoidance stress (WAS) has been shown to result in altered gut microbiota and acute small intestinal inflammation. These mice presented intestinal erythema, leukocyte infiltration, increased intestinal permeability, and increased mucosal expression of IL-17 and IL-6. Other inflammasome components that had their expression partially inhibited by WAS were ASC, caspase-1, IL-1β, and IL-18, while no significant impact was observed on NLRP3 expression. As observed for colitis and colitis-related CRC, non-stressed mice developed enteritis upon cohousing with WAS-exposed mice and probiotic therapy prior to WAS reduced intestinal inflammation and prevented WAS-mediated dysbiosis (115).

NLRP3-deficient mice have also been shown to be more susceptible to DSS-induced colitis, to have a dysbiotic microbiota, and to present altered colonic β-defensin expression and decreased antimicrobial capacity. Their neutrophils exhibited impaired chemotaxis and enhanced spontaneous apoptosis (116). Modulation of NLRP3 in the gut epithelium by high-fiber feeding has been suggested to contribute to intestinal homeostasis and protection from colitis through maintenance of a healthy microbiota and due to SCFA-mediated sensing by G-proteincoupled receptors GPR43 and GPR109A and IL-18 release (117). However, upon epithelial injury, members of the gut microbiota, such as *Enterobacteriaceae*, and in particular the pathobiont *P. mirabilis* induce NLRP3-mediated IL-1β release by Ly6Chigh monocytes, leading to intestinal inflammation (110).

The influence of inflammasome-mediated sensing of the microbiota goes beyond inflammatory processes in the gut. NLRP3, NLRP6, and inflammasome components ASC, caspase-1, and IL-18-deficiency-associated dysbiosis leads to exacerbated hepatic steatosis and inflammation through influx of TLR4 and TLR9 agonists into the portal circulation, which results in enhanced hepatic TNF-α expression and non-alcoholic steatohepatitis (NASH)/non-alcoholic fatty liver disease (NAFLD) progression. Cohousing experiments were sufficient to exacerbate NASH in WT mice, suggesting that intestinal dysbiosis may govern initial steps of systemic autoinflammatory disorders (118).

It has been shown that Pstpip2cmo mice, which are prone to develop osteomyelitis, present dysbiotic intestinal microbiota characterized by *Prevotella* outgrowth. Interestingly, these mice were protected from inflammatory bone disease and bone erosion when fed with high-fat diet (HFD), which had a marked impact on intestinal *Prevotella* reduction and significantly reduced pro-IL-1β expression in neutrophils. Antibiotic treatment was also efficient in reducing pro-IL-1β expression in Pstpip2cmo mice, but cohousing experiments and fecal microbiota transplantation from Pstpip2cmo mice failed to cause disease in WT mice. However, fecal microbiota transplantation from low-fat diet (LFD)-fed diseased Pstpip2cmo mice to young LFD-fed Pstpip2cmo mice by oral gavage promoted the expansion of *Prevotella*, and significantly accelerated the development of osteomyelitis, whereas transplantation of fecal microbiota from HFD-fed Pstpip2cmo mice to young LFD-fed Pstpip2cmo mice limited *Prevotella* outgrowth and significantly protected mice from developing osteomyelitis. Caspase-1/8-mediated processing of microbiota-induced pro-IL-1β was necessary to promote autoinflammatory disease in these susceptible mice (119).

Extraintestinal inflammatory processes have also been shown to be partially dependent on inflammasome-mediated sensing of the microbiota metabolite acetate. GF, antibiotic-treated, and GPR43-dificient mice are protected from joint inflammation upon injection of monosodium urate monohydrate (MSU) crystals. It was demonstrated that microbiota reconstitution or acetate administration to GF mice restored MSU crystals-induced inflammation and that GPR43 is at least partially necessary to adequate inflammasome assembly and IL-1β production in response to acetate (120). Therefore, one may suggest that inflammasomemediated sensing of both intestinal microbiota and their products contributes to control local and systemic inflammatory disorders, which should be considered when designing prophylactic and therapeutic applications.

## INFLAMMASOME AND TLR INTERACTIONS

Many studies have focused on the interactions between inflammasomes and TLRs. In fact, it has been shown that the enteric pathogens *E. coli*- and *Citrobacter rodentium*-induced TLR4/TRIF-dependent synthesis of caspase-11 and activation of NLRP3 inflammasome in macrophages (121) and that *Pseudomonas aeruginosa* infection of macrophages induced TLR-4/TRIF-dependent autophagy, which was attenuated by NLRC4/caspase-1-mediated cleavage of TRIF. Prevention of *in vivo* caspase-1-mediated cleavage of TRIF resulted in enhanced autophagy, reduced IL-1β production, and increased bacterial clearance (122). *L. monocytogenes* infection of macrophages was shown to induce TLR/IRAK1/IRAK4-dependent activation of NLRP3 (123, 124), which suggests that inflammasome–TLR interactions may have an important role in the bacterial recognition.

It has been also demonstrated that Clathrin-mediated endocytosis followed by TLR8- and TLR7-mediated recognition of HIV and HCV in monocytes and macrophages resulted in NLRP3 activation, which was independent of type I IFN production (125). Moreover, dendritic cells were shown to express higher levels of NLRP3 in the steady-state condition compared to macrophages and to secrete substantial amounts of mature IL-1β upon stimulation with TLR ligands independently of P2X7 signaling (126). On the other hand, chronic TLR stimulation by LPS has been shown to dampen NLRP3 activation through IL-10 induction (127), and TLR2/TLR4 engagement resulted in upregulation of plasminogen activator inhibitor type 2 (PAI-2), a serine protease inhibitor, which culminated in the stabilization of the autophagic protein Beclin 1 to promote autophagy, reduction of mitochondrial reactive oxygen species, NLRP3 protein level, and pro-IL-1β processing (128).

Although increasing evidence suggests that both TLRs and inflammasomes play an important role in promoting host– microbiota communication, studies regarding the interactions of these molecules in the context of the gut microbiota are still lacking and will further improve our knowledge on how commensal microorganisms and the immune system cooperate in the modulation of inflammatory and autoimmune diseases.

#### REFERENCES

## CONCLUDING REMARKS

The dynamic interactions that have been established between mammalian hosts and commensal microorganisms over millennia of coevolution resulted in the development of a wellstructured immune system, which controls pathogenic infections while tolerates a highly diverse microbiota. Innate sensing of the gut microbiota through TLRs and inflammasomes contributes to intestinal homeostasis by stimulating the development/function of both regulatory and inflammatory cells and by promoting proliferation of IECs, epithelial integrity, mucus secretion, and containment of opportunistic infections. It has become clear that disturbances in the fine-tuning that governs host–microbiota interactions may lead to both local and systemic inflammatory and autoimmune diseases. In this review, we bring many examples of the dualistic role played by the gut microbiota in both promoting and regulating inflammation and autoimmunity. We believe that a better understanding of mechanisms involved in these complex interactions between host and commensals will further improve common prophylactic and therapeutic applications.

#### AUTHOR CONTRIBUTIONS

AI, CM, and RA wrote the manuscript. NC and RA reviewed the manuscript.

#### ACKNOWLEDGMENTS

We gratefully acknowledge funding provided by CNPq and Sao Paulo State Research Funding Agency (FAPESP) (12/02270-2). RA, AI, and CM are recipients of FAPESP fellowships.

a protein linked to neurodegenerative disorders. *Sci Signal* (2015) **8**(376):ra45. doi:10.1126/scisignal.2005965

the balance of T cells in a mouse model of arthritis. *J Clin Invest* (2008) **118**(1):205–16. doi:10.1172/jci32639

commensally induced pyroptosis. *J Immunol* (2013) **190**(11):5676–88. doi:10.4049/jimmunol.1201592

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Ignacio, Morales, Câmara and Almeida. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Serum Cytokine Signature That Discriminates *Helicobacter pylori* Positive and Negative Juvenile Gastroduodenitis

Svetlana F. Khaiboullina1 , Sayar Abdulkhakov 1, 2, Alsu Khalikova2 , Dilyara Safina1 , Ekaterina V. Martynova1 , Yuriy Davidyuk 1 , Felix Khuzin2 , Rezeda Faizullina2 , Vincent C. Lombardi 1, 3, Georgi V. Cherepnev 1, 4 and Albert A. Rizvanov 1 \*

*1 Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia, 2 Kazan State Medical University, Kazan, Russia, 3 Nevada Center for Biomedical Research, Reno, NV, USA, 4 Department of Clinical Laboratory Diagnostics, Kazan State Medical Academy, Kazan, Russia*

#### *Edited by:*

*Ryo Inoue, Kyoto Prefectural University, Japan*

#### *Reviewed by:*

*M. Victoria Delpino, National Scientific and Technical Research Council (CONICET), Argentina Gustavo Adolfo Romero-Perez, Kyoto Institute of Nutrition and Pathology, Japan*

> *\*Correspondence: Albert A. Rizvanov<EMAIL_ADDRESS>
> > *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Microbiology*

*Received: 10 September 2015 Accepted: 15 November 2016 Published: 15 December 2016*

#### *Citation:*

*Khaiboullina SF, Abdulkhakov S, Khalikova A, Safina D, Martynova EV, Davidyuk Y, Khuzin F, Faizullina R, Lombardi VC, Cherepnev GV and Rizvanov AA (2016) Serum Cytokine Signature That Discriminates Helicobacter pylori Positive and Negative Juvenile Gastroduodenitis. Front. Microbiol. 7:1916. doi: 10.3389/fmicb.2016.01916* Gastroduodenitis caused by *H. pylori*, often acquired in early childhood, is found in about 50% of the adult population. Although *H. pylori* infections can remain asymptomatic, its virulence factors usually trigger epithelial vacuolization and degeneration, loss of microvilli, disintegration of cytoplasm, and leukocyte accumulation. It is believed that leukocyte infiltration is driven by cytokines produced locally in infected tissue. However, so far little is known about changes in serum cytokines in juvenile patients infected with *H. pylori*. Serum cytokine profiles were analyzed in 62 juvenile patients diagnosed with gastroduodenitis using the Bio-Plex multiplex assay. *H. pylori* infection was confirmed in 32 patients, while 30 patients were *H. pylori*-free. Cytokines CXCL5 and CXCL6, potent neutrophil chemoattractants, were upregulated in all patients diagnosed with gastroduodenitis. Serum levels of IL8, a prototype neutrophil attractant, remained unchanged in subjects with gastroduodenitis relative to controls. Therefore, our data suggest that CXCL5 and CXCL6 play a role in directing neutrophil trafficking into inflamed gastroduodenal tissue. In addition, the CCL25/GM-CSF ratio differed significantly between *H. pylori*-positive and -negative juveniles. Further, study is needed to evaluate the role of CCL25 and GM-CSF in the pathogenesis of the different etiologies of gastroduodenitis.

Keywords: *H. pylori*, virulence factors, cytokines, gastroduodenitis

## INTRODUCTION

Helicobacter pylori (H. pylori) was identified in 1982 and suggested to be a causative agent for gastritis and stomach ulcers (Marshall and Warren, 1984). This helix-shaped gram-negative bacterium colonizes gastric mucosa and persists as a chronic infection (Marshall et al., 1985a; Morris and Nicholson, 1987). H. pylori is one of the most common gastrointestinal infections, being found in about 50% of the adult population (Sipponen et al., 1996; Kosunen et al., 1997). The majority of H. pylori infections remain asymptomatic, with only 15% of carriers developing symptoms (Atherton, 1998; Ernst and Gold, 2000). Infection, often acquired in early childhood, has been shown to be associated with poor hygiene and impoverished living conditions (Malaty and Graham, 1994; Kivi et al., 2003; Konno et al., 2005; Dattoli et al., 2010). It is believed that H. pylori is transmitted via fecal-oral or oral-oral routes (Goh et al., 2011).

During gastric epithelium colonization, H. pylori establishes a persistent infection within the mucus layer without crossing the epithelial barrier (Noach et al., 1994). Infection is histologically characterized by surface epithelial degeneration, inflammation, and leukocyte infiltration into the gastric mucosa (Bodger and Crabtree, 1998; Peek et al., 2010).

H. pylori virulence factors are the main cause of tissue damage, and include vacuolating cytotoxin (vacA), cytotoxin associated gene A (cagA), and neutrophil-activating protein (HP-NAP; Atherton, 1998; Cellini and Donelli, 2000; Fu, 2014). Binding of VacA to gastric cells triggers epithelial vacuolization and degeneration, loss of microvilli, and cytoplasmic disintegration (Goodwin et al., 1986; Papini et al., 1994; Garner and Cover, 1996; Smoot et al., 1996). Animal studies have demonstrated that purified vacA toxin causes gastric epithelial damage with little effect on inflammatory leukocyte infiltration (Telford et al., 1994; Ghiara et al., 1995). In addition, cagA has been shown to be strongly associated with development of local inflammation and expression of pro-inflammatory cytokines (Peek et al., 1995). Moreover, it has been suggested that the cagA gene and nearby sequences code for proteins that act synergistically and promote production and secretion of pro-inflammatory cytokines (Tummuru et al., 1995; Censini et al., 1996), and that the virulence factor HP-NAP promotes neutrophil adhesion, chemotaxis, and activation (Satin et al., 2000). The combined effects of these virulence factors is inflammation of local tissue caused by damage to gastric epithelial cells, and activation of pro-inflammatory cytokine production.

Histologically, infiltration of gastric tissue by leukocytes is a hallmark of H. pylori infection. In tissue biopsies collected from patients infected with H. pylori both neutrophil infiltration (Kamada et al., 2006; Jaramillo-Rodríguez et al., 2011; Xu et al., 2012) and increased infiltration of CD4+ T helper lymphocytes in the lamina propria (D'Elios et al., 1997a) have been reported. Further, in gastric mucosa, it has been demonstrated that H. pylori infection activates predominantly Th1-type immune responses (D'Elios et al., 1997a), and immunohistochemically analyses of gastric biopsies have revealed an increased presence of CD8+ lymphocytes and macrophages (Bedoya et al., 2003). Animal models established that the early stage of infection is marked by neutrophil infiltration (Rossi et al., 2000). As infection progresses, a drop in neutrophil counts is followed by increased tissue infiltration with mononuclear leukocytes, mostly CD3+ lymphocytes. Initially scattered, lymphocyte infiltrates organize into small patches in the corpus and antrum of stomach (Rossi et al., 2000; Sepulveda and Patil, 2008). Later, the appearance of CD4+ and CD8+ lymphocytes in the periglandular area and beneath the basal lamina correlates with histological signs of gastric epithelial damage. Eventually, leukocyte infiltrates became organized in follicles containing CD21+, CD4+, and CD3+ lymphocytes (Rossi et al., 2000). Furthermore, the appearance of neutrophils at late stage leukocyte infiltration suggests active chronic gastritis. Together, these data indicate that initially H. pylori infection causes neutrophil infiltration of gastric mucosa, then as infection progresses neutrophils are replaced by lymphocytes. This leukocyte infiltration is the main cause of gastric epithelial damage in H. pylori infected tissue.

Exposure of gastric epithelium to H. pylori results in the production of a number of cytokines that stimulate migration of immune effector cells into inflamed tissue, including upregulation of IL8, CCL5, CCL3, IFN, IL10, IL12p40, and IL18 (Crabtree et al., 1994, 1995a,b; Yamaoka et al., 1996, 1998; Karttunen et al., 1997; Park et al., 2001; Dzierzanowska-Fangrat et al., 2008). Studies using animal models have demonstrated that the early stage of H. pylori infection is characterized by increased expression of IL1, IL8, IL6, and TNF-α in gastric mucosa (Harris et al., 2000; Rossi et al., 2000). Then, as the disease progresses, IL8 expression declines, while IL1, IL6, and TNF-α remain elevated (Rossi et al., 2000; Harris et al., 2000). It has been suggested that at late stages of the disease, there is a shift toward Th1 immunity, involving cytokines such as IFNγ and IL12 (D'Elios et al., 1997a; Haeberle et al., 1997; Pellicanò et al., 2007), and that persistent activation of the Th1 immune response is a cause of tissue damage in H. pylori infection (D'Elios et al., 1997b; Smythies et al., 2000). A combination of transcriptional analysis of tissue biopsies and histological findings has provided most of the information about cytokine activation of H. pylori infection. However, there is limited knowledge of serum cytokine expression in children infected with H. pylori, since the majority of data is from adult populations (Bayraktaroglu et al., 2004; ˘ Mehmet et al., 2004; Abdollahi et al., 2011; Eskandari-Nasab et al., 2013). To date, for the most part, cytokines studies have been limited to the Th1 or pro-inflammatory class. This is unfortunate, as H. pylori infection often occurs during early childhood establishing a lifelong chronic infection. Understanding cytokine expression at the initial stages of infection will identify early markers, and improve disease diagnosis.

Here, we present data on cytokine activation in serum of children with gastroduodenitis. Sixty-two juveniles with gastroduodenitis were included in this study, with 30 having a diagnosis of H. pylori infection. Regardless of the presence or absence of H. pylori pathogenicity markers, there was upregulation of the potent neutrophil chemoattractants, CXCL5, and CXCL6. However, serum levels of IL8, a prototype neutrophil attractant, were not statistically different between diagnoses. Therefore, our data suggest that a novel set of chemokines, CXCL5, and CXCL6, play a role in neutrophil trafficking into inflamed gastroduodenal tissue. Further, an intriguing observation was that the CCL25/GM-CSF ratio differed significantly between H. pylori positive and negative children with gastroduodenitis.

## MATERIALS AND METHODS

### Subjects

Sixty-two patients (24 boys, 38 girls; age 14.0 ± 2.1) hospitalized in the Children's Republican Clinical Hospital (Kazan, Russia) with a diagnosis of gastritis and duodenitis were recruited for this study. Initial diagnosis of H. pylori infection in 30 of the patients was based on clinical presentation and upper GI endoscopy. The presence of H. pylori was confirmed by urea breath test and PCR.

Biopsies were collected from each patient during upper GI tract endoscopy: two from antral part of the stomach along the major and minor curvatures, and 2–3 from the body of the stomach, along the major and minor curvatures. In addition, stomach biopsies were collected from three controls, who were found to be negative for any gastroduodenal pathology by diagnostic upper GI tract endoscopy. Biopsies were used for both PCR analyses and histological studies.

Serum samples were collected from all 62 juveniles with gastritis and duodenitis, as well as from 20 age- and sex-matched controls. All controls were negative for symptoms of upper GI tract infection or gastritis. Serum samples were stored at −80◦C. The Ethics Committee of Kazan State Medical University approved this study (N6, 06.25.2012) and informed consent was obtained from the legal guardian of each study subject, in accordance with the Declaration of Helsinki and the article 20, Federal Law "Protection of Health Right of Citizens of Russian Federation" (N323-83, 11.21.2011).

## Urea Breath Test

Breath ID Hp (Exalenz, USA) was used to confirm H. pylori infection.  After 10 min, exhaled air is collected and tested for the presence of 13CO2, which indicates H. pylori infection.

## PCR Detection of *H. pylori*

DNA was extracted from biopsy tissue using the Helicopol Kit (Lytech, Moscow). H. pylori positive biopsies were analyzed by PCR for a pathogenicity marker profile using the Helicopol II Kit (Lytech, Moscow). Briefly, 2 µl total DNA was mixed with 4 µl 10x PCR buffer, 2 µl 25 mM MgCl2, 0.5 µl (100 pmol) each of primers, 40.7 µl distilled water, and 0.3 µl (2.5 U) Taq DNA polymerase. The reaction mixture was then subjected to 35 cycles, each consisting of 30 s at 94◦C, 30 s at 50◦C, and 2 min at 72◦C. PCR products were analyzed on a 1% agarose gel.

## Cytokine Analysis

Serum cytokine levels were analyzed using a Bio-Plex Pro Human Chemokine Panel (40-Plex; Bio-Rad, Hercules, CA), a multiplex magnetic bead-based antibody detection kit.

## Immunohistochemical Analysis

After initial analysis, there were surplus clinical diagnostic biopsy specimens from 21 H. pylori positive and 13 negative cases. These were used for immmunohistochemical analysis. Punch biopsies were fixed in 4% paraformaldehyde for 4 h at 4◦C, and then cryoprotected with 30% sucrose in PBS. Immunohistochemical staining was performed on 5 µm thick sections. Slides were deparaffinized with xylene and rehydrated through a graded alcohol series. Tissue morphology was evaluated by light microscopy using hematoxylin-eosin staining. Additionally, Alcian blue (pH 2.5) and periodic acid Schiff (PAS) staining were performed to detect the presence of sialomucins.

### Statistical Analysis

Statistical analysis was performed using the STATISTICA 7.0 Software Package (StatSoft, Tulsa, OK) and the IBM SPSS Statistics 20 Software Package (IBM Corp, Armonk, NY, U.S.). Data are presented as the median (5–95% range) for continuous variables. Differences between independent study groups were tested by non-parametric methods. To address type 1 error, Kruskal–Wallis ANOVA by Ranks test for multiple independent samples was followed by the multiple comparison (all pairs) nonparametric post-hoc Steel-Dwass test Nonparametric multiple comparisons were made [recalculated and confirmed] by the Steel-Dwass all pairs test using JMP R 13.0.0 Software (SAS Institute Inc, USA). Differences were considered significant at P < 0.05. Jonckheere's non-parametric trend test was performed to compare three group medians when they were arranged in order. Cytokine profiles between subject groups were differentiated using forward stepwise discriminant function analysis.

## RESULTS

## Patients

Sixty-two patients (24 boys and 38 girls) were admitted to the Children's Republican Clinical Hospital (Kazan, Russia) with a diagnosis of gastritis and duodenitis. The main symptoms were pain in the epigastric area, vomiting, nausea, headache, and fatigue. Biopsy samples collected from the antrum of the stomach were analyzed for the presence of H. pylori antigens. Thirty samples were positive for H. pylori and 32 samples negative. The surplus biopsy tissue from 21 positive and 13 negative juveniles (of the 62 enrolled in the study) was used for histological studies.

Three H. pylori positive biopsies had massive leukocyte infiltration of the lamina propria (**Figure 1**) with moderate incomplete metaplasia of the epithelium (**Figure 2**). Moderate lymphocyte infiltration of the lamina propria was found in 11 positive biopsies, with four having moderate epithelial metaplasia. Finally, four had moderate lymphocyte infiltration of the lamina propria with no sign of metaplasia. Based on histological evaluation, diagnosis of chronic atrophic gastritis was established in 9 (42.9%) of the H. pylori positive biopsies.

Histological analysis of the 13 H. pylori negative biopsies revealed epithelial metaplasia in eight biopsies, with lymphocyte infiltration in five cases. Two biopsies had lymphocyte infiltration and metaplasia. The remaining two biopsies had no sign of lymphocyte infiltration or metaplasia. Diagnosis of chronic atrophic gastritis was established in 6 (46.2%) of the H. pylori negative biopsies. The gastritis activity was graded in all biopsies according to the Histological Division of the Sydney System and "Up-Dated Sydney System" (Tytgat, 1991; Dixon et al., 1997; **Table 1**). This classification determines the activity grade based on the presence of polymorphonuclear leucocytes in combination with mononuclear inflammatory infiltrate, intestinal metaplasia, atrophy, and detection of H. pylori organisms. H. pylori positive biopsies were characterized by a higher grade of chronic inflammation compared to H. pylori-free biopsies (**Table 1**).

All patients were examined by gastroduodenoscopy. Hyperemia and edema were detected in the gastric mucosa

FIGURE 1 | Histological sections of three representative biopsies. Gastric biopsy sections (3–5 µm) of control (A), and *H. pylori* positive (B) and *H. pylori* negative (C) gastroduodenitis cases were deparaffinized and stained with hematoxylin and eosin (H&E). The gastric epithelium phenotype of the *H. pylori* positive juvenile (B) resembles the phenotype of colonic epithelium, characterized by multiple intracytoplasmic mucin droplets of varying sizes and shapes (solid arrow), and the absence of a brush border (dashed arrow). H&E; x100; Bar represents 20 µm.

of some, but not all, subjects. No visible changes were observed in the esophagus. Erosion with fibrin deposits, hyperemia, and edema were detected in 3 (14%) of H. pylori positive subjects. Five (16.1%) H. pylori positive cases were characterized by duodenal bulbar deformity with multiple scars, hyperemia and edema, with no deformation, or scarring observed in H. pylori negative stomachs. Two (6.4%) H. pylori positive stomachs had multiple ulcers, while all H. pylori negative patients were all ulcer-free. Overall, H. pylori positive stomachs were characterized by pronounced histological abnormalities compared to H. pylori negative stomachs.

## Cytokine Profile

A total of 40 cytokines were analyzed in the serum of all subjects (**Table 2**). Children with gastroduodenitis had similar profiles, with the cytokine activation profile in H. pylori positive serum closely resembling H. pylori negative. For example, significant upregulation of CXCL2, CCL1, IL2, CCL7, CCL22, CXCL16, and CXCL12 was detected in both groups. Interestingly, the serum level of a major chemoattractant for neutrophils, IL8, did not differ significantly between patients with gastroduodenitis and controls. However, serum levels of CXCL5 and CXCL6, chemoattractant factors for neutrophils, were significantly upregulated in sera from all H. pylori positive juveniles, and not linked to the presence of neutrophil infiltration in biopsied tissue. Although serum levels of TNF-α were significantly higher in both H. pylori positive and negative sera compared to controls (**Table 2**), concentrations of this cytokine were within the normal range for this age (Mózes et al., 2011).

Significantly increased levels of CCL15, CCL20, and MIF were detected in both H. pylori positive and negative serum compared controls. However, only MIF levels differed significantly between the two groups. In contrast, no changes in serum levels of CCL2, CCL3, CCL8, CCL11, CCL17, CCL19, CCL21, CCL23, CCL24, CCL26, CCL27, CXCL9, CX3CL1, IL6, IL8, IL10, and IFN-γ were found either between the two groups of patients with gastroduodenitis, or between juveniles with gastroduodenitis and the control group.

We compared cytokine activation in H. pylori positive patients with different histological presentations. The histological data was used to divide the patients into four groups; group one was characterized by severe lymphocyte and neutrophil infiltration of the lamina propria with moderate epithelial metaplasia, group two had moderate lymphocyte infiltration of the lamina propria and moderate epithelial metaplasia, group three had mild infiltration of lamina propria and mild epithelial metaplasia, and group four had no lymphocyte infiltration and no epithelial metaplasia. The pattern of cytokine upregulation was similar in all groups and was characterized by increased serum levels of chemoattractants for lymphocytes, monocytes, natural killer cells and dendritic cells such as CCL1, CCL22, CCL15, CXCL16, and CXCL12.

Next, forward stepwise discriminant function analysis was utilized to identify cytokines that differed between control and H. pylori positive and negative sera. Fourteen cytokines were selected to generate a classification matrix [model summary: Wilks' lambda = 0.15498; F(28, 102) = 5.6106, p < 0.0001, IL4, IL8, CCL3, CCL1, CCL7, IFNγ, CXCL12, CCL2, CXCL10,

TABLE 1 | Histological examination of the gastric biopsies from *H. pylori* positive and negative gastroduodenitis cases.

CCL23, MIF, TNF-α, CXCL1, and CXCL9]. Discriminant analysis revealed greater differences between patients with gastroduodenitis (both H. pylori positive and negative) and controls (Squared Mahalanobis distances 34.32) than between H. pylori positive and negative patients (Squared Mahalanobis distances 2.56). Furthermore, the 14 cytokine-based classification matrix yielded 100% correct predictions for controls (predicted classifications vs. observed classifications in the classification matrix), with lower percentages of correct predictions for cases that were H. pylori positive (80%) and H. pylori negative (76.66%).

In Crohn's disease altered expression of GM-CSF and CCL25 has been suggested to play a role in the pathogenesis of inflammatory gastrointestinal disease (Samson et al., 2011). Therefore, we sought to determine whether these two cytokines were involved in the pathogenesis of H. pylori-related gastroduodenitis. Serum levels of GM-CSF were significantly upregulated in H. pylori negative subjects, while in H. pylori positive subjects, GM-CSF levels were similar to controls (**Table 2**), and significantly lower (∗P < 0.05; **Table 2**) than H. pylori-free serum. In addition to increased GM-CSF levels, H. pylori negative serum was characterized by significantly lower levels of CCL25 (**Table 2**), suggesting bidirectional activation of these cytokines in negative serum. To further analyze the activation pattern of these cytokines in H. pylori negative and positive serum, we compared the CCL25/GM-CSF ratio in three independent study groups using Kruskal–Wallis ANOVA by Ranks test (P-level = 0.0083), followed by post-hoc Jonckheere's non-parametric trend test for multiple comparisons (P-level = 0.006; **Figure 3**). The CCL25/GM-CSF ratio differed significantly between each group. H. pylori positive and negative juveniles were positioned on either side of controls (**Figure 3**), suggesting that the CCL25/GM-CSFratio reflects an essential difference in gastroduodenitis pathogenesis with H. pylori positive and negative stomach having distinct forms.

## DISCUSSION

H. pylori infection is often acquired early in childhood (McCallion et al., 1996; Suerbaum and Michetti, 2002; Tkachenko et al., 2007). This bacterium colonizes gastric mucosal epithelium, establishes a chronic infection (Marshall et al., 1985b; Morris and Nicholson, 1987), and once established, releases numerous virulence factors causing apoptosis and vacuolization of the gastric epithelium, and functional disruption of the gastric epithelial barrier (Goodwin et al., 1986; Papini et al., 1994; Garner and Cover, 1996; Smoot et al., 1996).

Currently, little is known about systemic activation of cytokines in children infected with H. pylori. The serum cytokine profile of infected children suggests a strong activation of chemoattractants for mononuclear leukocytes (**Table 2**). For example, in H. pylori positive gastroduodenitis we demonstrate increased serum levels of chemoattractants for mononuclear lymphocytes, such as CXCL10, CCL22, and CXCL16 (Taub et al., 1996; Andrew et al., 1998; Huang et al., 2008). This observation is supported by histological examination of biopsies, where increased leukocyte infiltration was detected (**Table 1** and **Figure 1**). Interestingly, there were no differences in the serum cytokine profiles of patients with distinct histological presentations suggesting that tissue pathology is determined by in situ cytokine activation, which is not reflected in circulating cytokine levels. Serum levels of the prototype neutrophil chemoattractant IL8 (Gessler et al., 2004), remained unchanged in juveniles with gastroduodenitis. Bayraktaroglu et al described the same phenomenon in adults where IL-8 serum levels in H. pylori positive cases did not differ from controls (Bayraktaroglu et al., 2004 ˘ ). However, increased levels of IL8 transcripts in tissue have been documented in H. pylori patients (Yamada et al., 2013; Nagashima et al., 2015), suggesting that upregulation of IL-8 may be a local characteristic of inflammation of gastrointestinal tissue. The molecular mechanisms regulating systemic neutrophil migration into inflamed tissue remains to be determined. Here, we demonstrate upregulation of CXCL5 and CXCL6, potent chemoattractants for neutrophils (Territo et al., 1989; Chertov et al., 1996; Mei et al., 2012) in serum from juveniles with gastroduodenitis. CXCL5, constitutively expressed by enterocytes, coordinates with CXCR2 the transmigration of neutrophils (Mei et al., 2012). Increased numbers of CXCL6 positive mucosal cells have been observed in Crohn's disease biopsies (Yamada et al., 2013), where, upon activation, upregulation of CXCL6 is more sustained than IL8 (Wuyts et al., 2003). It has been suggested that CXCL6 could play a role in supporting chronic inflammation by facilitating neutrophil migration at a late stage of infection (Wuyts et al., 2003). In this study, we present novel data on the upregulation of CXCL5 and CXL6 in serum during gastroduodenitis. Serum levels of these cytokines did not differ between H. pylori positive and negative children with gastroduodenitis, suggesting that upregulation of CXCL5 and CXCL6 is a tissue-specific response to inflammation, and not driven by a specific pathogen.

#### TABLE 2 | Serum cytokine profile of children diagnosed with *H. pylori* positive and negative gastroduodenitis.

*P—significance between gastroduodenitis group and healthy control, Steel-Dwass test.*

\**P—significance gastroduodenitis groups, Steel-Dwass test.*

CXCL6 is a neutrophil chemoattracting factor produced by endothelial cells and macrophages exposed to IL1β or LPS (Wuyts et al., 2003). Additionally, transcriptional activation of both CXCL6 and CXCL5 has been demonstrated in cells stimulated with IL17 (Ruddy et al., 2004; Numasaki et al., 2005). Here, we show upregulation of IL17 in H. pylori positive juveniles suggesting that IL17 activation of epithelial cells and local macrophages causes upregulation of CXCL5 and CXCL6, which in turn promotes neutrophil chemotaxis into gastric tissue. It should be noted that CXCL6 synergy with MCP1 facilitates neutrophil chemotaxis (Gijsbers et al., 2005). Therefore, neutrophil chemotaxis may result from a combined action of several of cytokines.

The most intriguing observation made in this study was that GM-CSF and CCL25 may play a role in the pathogenesis of gastroduodenitis, and that changes in serum levels of GM-CSF and CCL25 may reflect the host's reaction to disease. Analysis of the CCL25/GM-CSF ratio indicates that H. pylori positive and negative gastroduodenitis are unrelated clinical entities. Our data support the observation made by Samson et al. suggesting a role for GM-CSF and CCL25 in the pathogenesis of inflammatory gastrointestinal disease (Samson et al., 2011). These authors demonstrated that CD patients with high serum level of GM-CSF neutralizing antibodies had increased number of iliac epithelial cells expressing CCL25. Local upregulation of CCL25 has been shown to facilitate iliac inflammation by stimulating CCR9-driven T lymphocyte migration. Our data provide further evidence for a role of both CCL25 and GM-CSF in the pathogenesis of inflammatory gastrointestinal diseases. Although our analysis revealed no correlation between the CCL25/GM-CSF ratio, histological presentation, and localization of gastrointestinal inflammation, we believe further study should be conducted to fully understand the function of CCL5 and GM-CSF in the pathogenesis of inflammatory gastrointestinal disease.

We have demonstrated upregulation of serum CXCL5 and CXCL6 in subjects diagnosed with gastroduodenitis, whereas serum levels of the neutrophil attractant, IL8, did not differ from controls. Therefore, we suggest that neutrophil accumulation in tissue in children with gastroduodenitis is directed by a distinct set of chemokines including CXCL5 and CXCL6. Additionally, we present the first evidence for a potential role for CCL25 and GM-CSF in the pathogenesis of gastroduodenitis, and that the CCL25/GM-CSF ratio can be utilized to for the discrimination of gastroduodenitis caused by H. pylori from gastroduodenitis due to other causes. Further study, using animal model for H. pylori gastroduodenitis is needed to determine the roles of CCL25 and GM-CSF in the pathogenesis of gastroduodenitis.

## AUTHOR CONTRIBUTIONS

SK, AR, VL, EM—cytokine profile analysis, SA, AK, DS, RF clinical examination of patients, FK—morphology, YD and GC biostatistics.

## ACKNOWLEDGMENTS

The work was performed according to the Russian Government Program of Competitive Growth of Kazan Federal University and subsidy allocated to Kazan Federal University for the state assignment in the sphere of scientific activities. Some of the experiments were conducted with support of the Interdisciplinary center for collective use of Kazan Federal University supported by Ministry of Education of Russia (ID

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Khaiboullina, Abdulkhakov, Khalikova, Safina, Martynova, Davidyuk, Khuzin, Faizullina, Lombardi, Cherepnev and Rizvanov. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# **An** *in silico* **approach reveals associations between genetic and epigenetic factors within regulatory elements in B cells from primary Sjögren's syndrome patients**

#### *Edited by:*

*Gayane Manukyan, National Academy of Sciences of the Republic of Armenia, Armenia*

#### *Reviewed by:*

*Rudra Bhowmick, Oklahoma State University, USA María-Julieta González, Universidad de Chile, Chile Gabriel J. Tobón, Fundación Valle del Lili, Colombia*

#### *\*Correspondence:*

*Yves Renaudineau, Laboratory of Immunology and Immunotherapy, Brest University Medical School Hospital, CHU Morvan, 2 Avenue Foch, BP824, Brest F29609, France<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

> *Received: 12 June 2015 Accepted: 11 August 2015 Published: 26 August 2015*

#### *Citation:*

*Konsta OD, Le Dantec C, Charras A, Brooks WH, Arleevskaya MI, Bordron A and Renaudineau Y (2015) An in silico approach reveals associations between genetic and epigenetic factors within regulatory elements in B cells from primary Sjögren's syndrome patients. Front. Immunol. 6:437. doi: 10.3389/fimmu.2015.00437* *Orsia D. Konsta1 , Christelle Le Dantec1 , Amandine Charras 1 , Wesley H. Brooks 2 , Marina I. Arleevskaya3 , Anne Bordron1 and Yves Renaudineau1,4 \**

*1 INSERM ESPRI, ERI29/EA2216, SFR ScInBioS, LabEx IGO "Immunotherapy Graft Oncology", Innovative Medicines Initiative PRECISESADS, Réseau épigénétique et réseau canaux ioniques du Cancéropole Grand Ouest, European University of Brittany, Brest, France, 2 Department of Chemistry, University of South Florida, Tampa, FL, USA, 3 Department of Rheumatology, Kazan State Medical Academy, Kazan, Russia, 4 Laboratory of Immunology and Immunotherapy, CHU Morvan, Brest, France*

Recent advances in genetics have highlighted several regions and candidate genes associated with primary Sjögren's syndrome (SS), a systemic autoimmune epithelitis that combines exocrine gland dysfunctions, and focal lymphocytic infiltrations. In addition to genetic factors, it is now clear that epigenetic deregulations are present during SS and restricted to specific cell type subsets, such as lymphocytes and salivary gland epithelial cells. In this study, 72 single nucleotide polymorphisms (SNPs) associated with 43 SS gene risk factors were selected from publicly available and peer reviewed literature for further *in silico* analysis. SS risk variant location was tested revealing a broad distribution in coding sequences (5.6%), intronic sequences (55.6%), upstream/downstream genic regions (30.5%), and intergenic regions (8.3%). Moreover, a significant enrichment of regulatory motifs (promoter, enhancer, insulator, DNAse peak, and expression quantitative trait loci) characterizes SS risk variants (94.4%). Next, screening SNPs in high linkage disequilibrium (*r* 2 *≥* 0.8 in Caucasians) revealed 645 new variants including 5 SNPs with missense mutations, and indicated an enrichment of transcriptionally active motifs according to the cell type (B cells *>* monocytes *>* T cells *≫*A549). Finally, we looked at SS risk variants for histone markers in B cells (GM12878), monocytes (CD14+) and epithelial cells (A548). Active histone markers were associated with SS risk variants at both promoters and enhancers in B cells, and within enhancers in monocytes. In conclusion and based on the obtained *in silico* results that need further confirmation, associations were observed between SS genetic risk factors and epigenetic factors and these associations predominate in B cells, such as those observed at the FAM167A–BLK locus.

**Keywords: Sjögren's syndrome, genetics, epigenetics, polymorphism,** *in silico***, histone modifications, B cells**

#### **Introduction**

Primary Sjögren's syndrome (SS) is a systemic autoimmune epithelitis affecting exocrine glands, such as salivary and lacrimal glands (1). The clinical manifestations of SS include dry mouth (xerostomia), dry eyes (keratoconjunctivitis sicca), systemic features, and patients have a 20- to 40-fold increased risk of developing lymphoma (2–4). Histological examination shows focal and peri-epithelial T and B cell infiltration plus macrophages in exocrine glands and parenchymal organs, such as kidney, lung, and liver (5). SS is characterized by the presence of circulating autoantibodies (Ab) against the sicca syndrome (SS)A/Ro and SSB/La ribonucleoprotein particles (6).

It is estimated that there are over 120 million single nucleotide polymorphisms (SNPs) in the human genome (NCBI dbSNP database, Build 143) and, among them, hundreds are disease risk variants for autoimmune diseases (AID) with the particularity that they are for the vast majority excluded from proteincoding regions (exon) and present within regulatory areas (7, 8). Regulatory SNPs control genes through an effect on (i) the transcriptional machinery when present within a gene regulatory region [promoter, enhancer, insulator (a gene regulatory element that blocks interaction between enhancers and promoters), and expression quantitative trait loci (eQTL)], (ii) the spliceosomal complex formation that controls intron excision, (iii) the activation of mRNA non-sense-mediated decay (NMD), and (iv) the control of messenger RNA stability through microRNA (3*′* -UTR). In SS, the list of genetic variations is growing with the particularity that the odds ratio (OR) is usually modest (OR *<* 1.5) with the exception of the HLA genes that have a significant OR (usually*>*2) (9). The associated risk genes analysis supports immunopathological pathways in SS, such as antigen presentation, cytokine signaling, and the NF-κB pathway (10). The characterization of regulatory SNPs in SS remains to be established.

In SS, several arguments support a role for epigenetic deregulation in disease initiation and progression (11, 12). The first clue was that two drugs, procainamide and hydralazine, induced SS in humans by blocking DNA methylation (13). Moreover, defects in DNA methylation characterize T cells, B cells, and salivary gland epithelial cells from SS patients (14–16), and such defects were associated with the expression of genes usually repressed by DNA methylation, such as transposons and miRNAs in salivary glands from SS patients (17, 18). Last, but not least, histone epigenetic markers and ribonucleoprotein post-translational modifications are immunogenic leading to autoAb production (14).

Accordingly, the aim of this work was to test the association between genetic and epigenetic determinants in SS. In the following, we pursue a two-staged analysis. First, we characterized a large panel of SS risk variants to reveal that they are predominantly present within regulatory elements. Second, we further explored the striking associations of those regulatory elements with cellular specificity and particularly in immune cells.

## **Materials and Methods**

#### **SS Genetic Risk Factors**

Data mining based on peer reviewed literature information (PubMed) and publicly available databases (centralgwas.org) served in the compilation of a list of 43 gene risk factors and their reported variants (*n* = 72) in SS (Table S1 in Supplementary Material) (19–49). The number of SS patients and controls were also reported as well as the OR average (95%), when available. The gene list used in this study was manually updated further to include gene function, SNP number (dbSNP database), and genomic location according to the human genome reference GRCh38. Genetic variants and their observed associations with clinical and functional phenotype were submitted to The National Center for Biotechnology Information (NCBI) ClinVar database1 . The gene list was tested with the FatiGO web interface AmiGO22 for functional enrichment.

#### **Functional/Regulatory Genome Annotation Data**

The variant effect predictor (VEP) tool3 was used to determine the location of the variants (exon, intron, 5*′* /3*′* -UTR, Up/Downstream genic sequence, and intergenic section) and their consequences [missense, non-coding transcript, splice donor variant, and target of non-sense-mediated mRNA decay (NMD)].

Assessment of the SNPs functional relevance was further completed by requesting Regulome DB V1.14 , and HaploRegV25 , data bases (50, 51) for promoter [RNA polymerase II (Pol II) binding], enhancer (H3K27Ac, H3K36me3, and/or H3K27me3 binding in the absence of Pol II binding), insulator [CCCCTCbinding factor (CTCF)], transcription factor (TF) binding, DNase peak, and eQTL.

#### **Linkage Disequilibrium**

Following SNP selection, the HaploRegV2 web portal was used to identify SNPs in linkage disequilibrium (LD, *R* 2 *≥* 0.80) in Europeans from the 1000 genome project using a maximum distance between variants of 200 kb in order to cover the enhancer elements (51).

#### **Statistical Analysis**

Pearson's Chi-squared test with Yate's continuity correction, when appropriate, was used to evaluate the significance of differences between the regulatory motifs and the histone chromatin immunoprecipitation (ChIP) experiments. A probability (*P*) of *<*0.05 was considered significant.

### **Results**

#### **Autoimmune-Related Genes Associated with SS**

A list of 43 SS-associated gene risk factors corresponding to 72 SNPs, referred to as SS risk variants, was extracted from the scientific literature (**Figure 1**). Among the risk factors, half (36/72) were associated with another AID (systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, inflammatory bowel disease, autoimmune thyroiditis disease, insulin-dependent diabetes, primary biliary cirrhosis, autoimmune hepatitis), allergy, infections, and cancer, including B/T cell lymphomas. This partial

1 http://www.ncbi.nlm.nih.gov/clinvar

2 http://amigo2.geneontology.org

3 http://www.ensembl.org 4

http://regulome.stanford.edu 5 http://www.broadinstitute.org

SS-associated risk variants were obtained from peer reviewed scientific literature, and their location determined. Next, they were tested for the

nucleotide polymorphisms (SNP) in high linkage disequilibrium (LD) was also assessed.

overlap suggests that both common and distinct genetic traits are present in SS and equally distributed.

#### **Variant Location**

Next, we used the VEP predictor tool in order to test the location of the different variants (**Figure 2**). Applied to the 72 SS risk variants, the VEP tools identified 4/72 (5.6%) exonic variants with missense mutations (IL17F, rs763780; MBL2, rs1800450; PTPN22, rs2476601; and TNFA1P3, rs2230926), and 40/72 (55.6%) intronic variants including an alternative splice donor variant (IRF5, rs2004640) and 4 variants that were predicted as targets of non-sense-mediated mRNA decay (NMD: ICA1, rs17143355; SLC25A40, rs10276819; STAT1, rs13005843; and TNIP1, rs6579837). Moreover, two 5*′* -UTR variants (CD14, rs2569190; and NCR3, rs11575837), one 3*′* -UTR variant (IL10, rs3024498), 12/72 (16.7%) upstream genic variants, 7/72 (9.7%) downstream genic variants, and 6/72 (8.3%) intergenic variants (*>*10 kb) were also observed.

#### **Regulatory Regions and DNA Binding Molecules**

We then used a combination of three tools based on information from the ENCODE program (VEP) and from both the ENCODE and the Roadmap Epigenome programs (RegulomDB and HaploReg v2) to determine whether SS risk variants are likely to be within promoters, enhancers, or insulators. These regulatory motifs were defined according to the available ChIP results from multi-cell analysis showing 21/72 (29.2%) promoters, 41/72 (56.9%) enhancers, and 5/72 (6.9%) insulators. Of particular note, within the four SNPs with missense mutations, one promoter and two insulators were detected (**Figure 3**). Moreover, 34/72 (47.2%) DNase hypersensitive regions (DNase peak) and 12/72 (16.7%) eQTL were recovered. Looking specifically at promoters and enhancers, data from ChIP experiments revealed that NFκB (*n* = 5), STATs (*n* = 3), and EGR-1 (*n* = 3) were predominant in promoters, and NF-κB (*n* = 3) in enhancers. For the remaining 5/72 (6.9%) SNPs, no regulatory functions were assigned which is significantly lower than the expected rate of 56.2% (*P <* 10*−*6 ) (50).

#### **Genes in High Linkage Disequilibrium**

In order to improve the analysis, we used the HaploReg v2 tool to include 645 new SNPs that were identified to be in high LD with the 72 annotated SNPs (**Table 1**). This tool identifies 34 new genes, including one microRNA (Mir4752), five SNPs with

missense mutations (rs1041981 in LTA; rs78957773 in MCCD1, rs2230539 in PKN1, and rs52817781 plus rs2233290 in TNIP1), and indicated an overall 15.8-fold enrichment of enhancers in human embryonic cells (H1; *P* = 0.0001) and 4.9-fold enrichment of enhancers in the GM12878 EBV-transformed lymphoblastoid B cell line (*P* = 0.0014). A significant enrichment of transcriptionally active sites, identified by DNAse enrichment analysis, was observed in the SNP set with a 21-fold increase in GM12878

**H3k36me3, and/or H3k7me3 binding), and insulators (CTCF binding)**.

lymphoblastoid B cells (*P <* 10*−*6 ), 10.8-fold increase in CD14+ monocytes (*P* = 0.00002), 9.2-fold increase in CD20+ B cells (*P* = 0.001), 8.2- to 8.5-fold increase in T cells (*P <* 0.005), and 5.7-fold increase in the A549 epithelial cell line (*P* = 0.005). Moreover, gene ontology biological process analysis (AmiGO2) identified "regulation of immune response" (*P* = 2.16 *×* 10*−*18 and 9.2 *×* 10*−*17), "positive regulation of cytokine production" (*P* = 1.56 *×* 10*−*11 and 1.22 *×* 10*−*13), "response to molecule of

#### **TABLE 1 | Gene risk factors associated with primary Sjögren's syndrome (SS)**.

#### **SS gene risk factors extracted from the literature**

BAK1, BCL2, BLK, C4A, CCL2, CD14, CD40, CD40LG, CHRM3, CXCR5, EBF1, FAM167A, GTF2I, HLA DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, ICA1, IKBKE, IL10, IL12A, **IL17F**, IL21, IRF5, LILRA3, **LTA**\*, **MBL2**, NCR3, NFKB1, OR2B11, **PKN1**\*, **PTPN22,** SLC25A40, STAT1, STAT4, TNF, **TNFA1P3**, TNFSF13B, **TNIP1**\*, TNPO3, TNFSF4, Trim21

#### **New genes in high linkage disequilibrium**

ABCB1, AC053545.3, ADAD1, AF213884.2, APBB3, ATP6V1G2, ATP6V1G2-DDX39B, BTNL2, C6orf10, CCL11, CCL7, CTD-2049J23.2, DBF4, GGNBP1, HCG23, HLA-DPA1, IL2, KIAA1109, **MCCD1**\*, Metazoa\_SRP, MIR4752, NDUFA2, NFKBIL1, PTGER1, RP11-10J5.1, RP11-356I2.2, RP11-356I2.4, RP3-527F8.2, RP5-998H6.2, RSBN1, RUNDC3B, SLC25A40, snoU13, XXbac-BPG254F23.7

*Bold missense mutations. \*Missense mutations detected by linkage disequilibrium.*

bacterial origin" (*P* = 4.9 *×* 10*−*7 and 6.2 *×* 10*−*9 ), and "cellular response to interferon" (*P* = 1.82 *×* 10*−*7 and 1.86 *×* 10*−*7 ) as the most significant functions associated with the SS gene risk factors tested alone or associated with the genes revealed by the LD analysis, respectively.

#### **Cell Type-Specific Analysis Revealed Activated Enhancer and Promoter Histone Markers at SS Risk Variants in B Cells**

To further explore cell type specific activation in promoters and enhancers at SS risk variants and according to the critical role played in the disease by epithelial cells, lymphocytes, and macrophages, we selected from the 18 ENCODE available cells: the human lung adenocarcinoma cell line A549 for epithelial cells, the GM12878 lymphoblastoid cells for B cells, and the peripheral blood CD14+ monocytes for macrophages. For these three cell types, we mapped SS risk variants to markers of active promoters (H3K4me2, H3K4me3, and H3K9Ac), and to markers of active enhancers (H3K36me3 and H3K4me1) (52). In addition, H3K27Ac was selected as a marker of activity, and H3K27me3 as an inactive marker of enhancers.

As shown in **Figure 4A** and with regards to the 21 promoter SS risk variants, the three active promoter markers (H3K4me2, H3K4me3, and H3K9Ac) were significantly enriched in B cells (GM12878) in contrast to the epithelial cells (A549) and monocytes (0.01 *< P <* 0.0006, Chi square with Yate's correction). The active marker H3K27Ac was enriched in B cells and monocytes in contrast to epithelial cells (*P* = 0.0001 and *P* = 0.02, respectively).

The same analysis was performed with the 41 enhancer SS risk variants (**Figure 4B**) revealing an enrichment of the enhancer active marker H3K36me3 in both B cells and monocytes in contrast to A459 cells (*P* = 0.02 and *P* = 0.005, respectively). The active marker H3K27Ac was enriched in B cells (*P* = 0.0001), and, although not significant, there is a trend for a monocyte enrichment in contrast to epithelial cells. In summary, these findings highlight the critical role of epigenetic factors in B cells to control both promoter and enhancer SS risk variants, and in monocytes to control enhancer SS risk variants.

#### **FAM167A–BLK Locus**