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Exosomal CagA from Helicobacter pylori aggravates intestinal epithelium barrier dysfunction in chronic colitis by facilitating Claudin-2 expression

Exosomal CagA from Helicobacter pylori aggravates intestinal epithelium barrier dysfunction in... Background: The chronic infection with Helicobacter pylori (H. pylori), especially cytotoxin-associated gene A-positive (CagA ) strains, has been associated with various extragastric disorders. Evaluating the potential impacts of virulence factor CagA on intestine may provide a better understanding of H. pylori pathogenesis such as colitis. The intestinal mucosal barrier is essential for maintaining its integrity and functions. However, how persistent C agA H. pylori coloni- zation influences barrier disruption and thereby affects chronic colitis is not fully understood. Results: Chronic colitis models of CagA H. pylori-colonized mice treated with 2% Dextran sulphate sodium (DSS) were established to assess the disease activity and pertinent expression of tight junction proteins closely related to mucosal integrity. The aggravating effect of CagA H. pylori infection on DSS-induced chronic colitis was confirmed in mouse models. In addition, augmented Claudin-2 expression was detected in C agA H. pylori infection conditions and selected for mechanistic analysis. Next, GES-1 human gastric epithelial cells were cultured with C agA H. pylori or a recombinant CagA protein, and exosomes isolated from conditioned media were then identified. We assessed the + − + Claudin-2 levels after exposure to CagA exosomes, CagA exosomes, and IFN-γ incubation, revealing that CagA H. pylori compromised the colonic mucosal barrier and facilitated IFN-γ-induced intestinal epithelial destruction through CagA-containing exosome-mediated mechanisms. Specifically, CagA upregulated Claudin-2 expression at the tran- scriptional level via a CDX2-dependent mechanism to slow the restoration of wounded mucosa in colitis in vitro. Conclusions: These data suggest that exosomes containing CagA facilitate CDX2-dependent Claudin-2 main- tenance. The exosome-dependent mechanisms of CagA H. pylori infection are indispensable for damaging the mucosal barrier integrity in chronic colitis, which may provide a new idea for inflammatory bowel disease (IBD) treatment. Keywords: CagA, Exosome, Colitis, Tight junction, Claudin-2, CDX2 Introduction Helicobacter pylori (H. pylori), a Gram-negative micro- aerophilic bacterium in the stomach, is one of the most ubiquitous pathogens as it colonizes more than half of the *Correspondence: xiaxiujuan2007@163.com; liuxiaoming26@163.com world’s population [1]. H. pylori infection is closely asso- Department of Gastroenterology, Third Xiangya Hospital of Central South University, 138 Tongzipo Road, Changsha 410013, China ciated with several gastric diseases, including gastritis, Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Guo et al. Gut Pathogens (2022) 14:13 Page 2 of 13 peptic ulcer disease, and gastric cancer [2]. Among the immunomodulatory effect in IBD since its DNA inhibits reported pathogenic factors of H. pylori, cytotoxin- proinflammatory cytokine production of dendritic cells, associated gene A protein (CagA) has been the focus of equilibrates Th17/Treg and Breg cell responses, and shifts attention. CagA is a specific virulence factor protein of macrophages toward M2 anti-inflammatory environ - H. pylori that enters host cells through the type IV secre- ments [27–30]. Intriguingly, when considering the exact tion system (T4SS). Once inside the cell, CagA triggers a pathogenic or protective effects of CagA on IBD or colitis variety of signal transduction pathways, induces changes at the molecular levels, the conclusions are lacking. For in cell morphology, and increases the risk of gastric dis- example, dextran sulphate sodium (DSS)-induced colitis eases [3, 4]. Moreover, increasing evidences show that was prominently deteriorated in CagA-transgenic mice + − CagA H. pylori strains other than C agA H. pylori are [31]. The biological basis for this effect regarding mucosal pathogens closely related to diseases outside the stom- barrier integrity remains unclear. ach [5, 6]. Deposited in renal tubules, CagA brings about Exosomes are extracellular vesicles derived from mul- strongly mucosal immune response in IgA nephropathy tivesicular bodies and are secreted by healthy cells, can- [7]. CagA H. pylori strains induce premature senescence cer cells, and host cells [32]. During infection, exosomes of extragastric cells, which may contribute to chronic released from host cells can convey pathogenic compo- skin diseases [8]. CagA seropositivity are highly corre- nents [33]. Exosomes are key regulators of cellular physi- lated with sarcopenia and low muscle quantity [9]. In the ological functions and pathogenesis [34]. A recent study context of CagA’s contribution to extragastric diseases, a reported that exosomes containing CagA were released large proportion of conundrums such as inflammatory from H. pylori CagA-expressing cells and could be bowel disease (IBD) remain to be fully elucidated. detected in blood circulation [35]. Moreover, exosomes Among the defensive mechanisms of the intestinal released from H. pylori-infected host gastric epithelial mucosa, an impaired epithelial barrier is the main path- cells can enter the bloodstream and aortic plaques, pro- ological factor that causes colitis, and it contributes to mote the formation of macrophage-derived foam cells, increased permeability by allowing increased antigen and accelerate the progression of atherosclerosis [36, 37]. penetration thereby initiating and propagating the dys- u Th s, exosome-carried CagA protein may be involved regulated response [10–13]. The normal intestinal bar - in the development of a variety of extragastric diseases rier consists of an intact layer of epithelial cells, which including IBD. are connected by a system of tight junction (TJ) strands To elucidate how H. pylori infection could affect IBD- [14]. TJs are composed of the occludin and claudin fam- associated chronic colitis via TJs, mouse models were ily members, which are transmembrane proteins, as well established, and in  vitro experiments were conducted in as TJ-associated proteins, such as Claudin-2 and ZO-1 the present study. CagA H. pylori infection was used to [15, 16]. Claudin-2 has been shown to induce cation- test the hypothesis that exosomal CagA from H. pylori selective channels in the tight junctions of epithelial cells, compromised the barrier integrity of the intestinal epi- and its expression is increased in IBD, leading to diar- thelium in colitis by facilitating Claudin-2 expression. rhea through the leakage flux mechanism [17, 18]. It was recently reported that Claudin-2 degradation enhances Results TJ barrier function in intestinal epithelial cells [19]. Spe- CagA H. pylori infection increases Claudin‑2 expression cifically, Claudin-2 expression is positively associated and aggravates DSS‑induced chronic colitis in C57BL/6 with inflammatory activity in patients with IBD, and plays mice multiple roles, such as targeting the vitamin D receptor, To determine the effect of H. pylori on the severity of in IBD pathogenesis [20, 21]. Another TJ protein, ZO-1, DSS-induced colitis, we first established a H. pylori- connects occludins and claudins to cytoskeletal actin, infected C57BL/6 mouse model under chronic inflam - and the loss of ZO-1 leads to increased intestinal per- mation conditions (Fig.  1A). Histopathological analysis meability [16]. ZO-1 preserves the intestinal barrier, and of the mouse gastric tissues of the mice after 8  weeks decreased intestinal inflammation has been observed [22, of H. pylori (CagA strain) infection showed positive 23]. To the best of our knowledge, the potential impli- Giemsa and silver staining for H. pylori in all the tested cations of TJ dysregulation in IBD pathogenesis remain animals, confirming that the mouse model of H. pylori elusive. infection was successful (Additional file  3: Figure S1). Certain clinical studies and systematic reviews have We then compared and analyzed the body weight and indicated the association between H. pylori exposure disease activity index (DAI) of mice with and without H. and reduced IBD risk [24–26]. The clinically observed pylori infection every three days after 2% DSS adminis- inverse association between H. pylori and IBD could be tration. The anatomical and histopathological features of partly explained by the fact that H. pylori might exert an the colon and spleen in each group were also evaluated. Guo  et al. Gut Pathogens (2022) 14:13 Page 3 of 13 Fig. 1 CagA H. pylori infection exacerbates DSS-induced chronic colitis in mice. A Schematic depiction of the experimental design for H. pylori (CagA strain) inoculation followed by chronic colitis models administrated 2% DSS. B, C Body weights (B) and disease activity index (DAI) changes (C) in different groups. Data from the second and third DSS-treatment cycles (7 day 2% DSS and 7 day diluted water) showed that H. pylori-infected mice had significantly less body weight loss and disease manifestation than control mice with no H. pylori infection in DSS-treated conditions. ***p < 0.001, compared to DSS group. Student’s t test for body weights, and χ test for DAI scores. D, E, F Colon lengths (D), spleen weights (E), microscopic appearance and H&E histological sections of the colon F in each group. Scale bars, 200 µm (200 ×) and 100 µm (400 ×). **p < 0.01, ***p < 0.001. Student’s t test was used for colon length and spleen weights, and the χ test was used for histological scores. G The relative expression of tight junction proteins ( TJs) in the colon of H. pylori and chronic colitis mice. The augmented Claudin-2 was observed in the H. pylori + DSS group in comparison with the DSS group. H The TJs protein expression of chronic colitis mice subgroups without H. pylori, with C agA H. pylori infection, + + − and with CagA H. pylori infection. Claudin-2 was significantly upregulated in CagA H. pylori-infected group compared with CagA H. pylori mice. All data were presented as means ± SD (n = 10) Guo et al. Gut Pathogens (2022) 14:13 Page 4 of 13 As expected, the DSS group showed significant weight colitis conditions (CagA negative status was identi- loss and higher DAI scores. In comparison with the nor- fied in Additional file  4: Figure S2). The colon histologi - mal control (NC) group, the mice with H. pylori infec- cal sections, colon lengths, and spleen weight verified tion alone (Hp group) displayed no significant changes that aggravated inflammation and intestinal injury were + − in weight or DAI scores. However, the mice pretreated detected in CagA H.pylori group compared to C agA with H. pylori infection exhibited more weight loss and H.pylori group (Additional file  5: Figure S3A, B, and manifestations than the DSS-treated group, and the more C). As for TJs tendency, claudin-2 levels in CagA H. intriguing finding was that these damaging effects of H. pylori group was markedly increased in comparison with − − pylori colonization on chronic colitis were obviously CagA H.pylori group. Intriguingly, CagA H.pylori was detected in the second and third 2% DSS cycles (Fig.  1B, not responsible for upregulated claudin-2 compared to C). chronic colitis mice subgroup without H. pylori infec- All the mice with DSS-induced colitis showed vary- tion (Fig.  1H). Collectively, CagA H. pylori infection ing degrees of shorter intestine, larger spleen weight, increases Claudin-2 expression and aggravates DSS- and loss of crypt and goblet cells with distinct mucosal induced chronic colitis. and submucosal infiltration of inflammatory cells, while H. pylori-pretreated mice showed significantly distinct CagA‑containing exosomes upregulate Claudin‑2 anatomical and histopathological abnormities (Fig.  1D, expression to compromise the intestinal mucosal barrier E, and F). The above data suggest a possible injuring role integrity in vitro of H. pylori in enhancing DSS-induced chronic colitis, To determine how CagA H. pylori infection could affect which is associated with incomplete regulation of the DSS-induced colitis, we hypothesized that CagA H. mucosal epithelium by TJs. Western blot analysis of the pylori exerted its function through CagA-containing expression of potential TJs was performed in the control exosome-mediated mechanisms. Typical exosomes were and experimental groups. Claudin-2 level was gradu- present in the conditioned media of GES-1 cells cul- ally increased in Hp and DSS group, reaching the peak tured with CagA H. pylori and were characterized by in Hp + DSS groups, as expected, while claudin-2 levels their specific biomarkers (HSP70, CD9, and CagA), size gradually increased in the Hp and DSS groups, peaking in distribution, and morphology (Fig.  2A, B, and C). These the Hp + DSS group. ZO-1 expression was substantially western blotting and electron microscopy results con- downregulated in the DSS groups, as expected, while the firm the uptake of exosomes by NCM460 cells, which is increasing tendency of ZO-1 in Hp + DSS group is not analogous to our previous immunofluorescence staining obvious. No changes in occludin were observed among results that the unique H. pylori pathogenic factor CagA the abovementioned groups (Fig. 1G). entered human GES-1 cells after they were incubated The trend of Claudin-2 expression might explain how with CagA H. pylori [37]. H. pylori exerts its aggravating effect in DSS-induced Compared to C agA H. pylori strain, western blot chronic colitis. Since the utilized bacterial in previ- and immunofluorescence showed that NCM460 ous study was C agA H.pylori strain, we tried our best cells exposed to exosomes from CagA H. pylori and to address if CagA, the strongest virulent factor for H. GES-1 coculture conditions directly augmented Clau- pylori, was involved in varied barrier function in chronic din-2 protein expression while depleting ZO-1 lev- + − colitis status. Both C agA H.pylori and C agA H.pylori els. Mucosal integrity and inflammatory condition were inoculated into mice followed by 2% DSS induced were characterized before and after an inflammatory (See figure on next page.) Fig. 2 Exosomes from GES-1 human gastric epithelial cells cultured with CagA H. pylori significantly disrupt the intestinal mucosal barrier meanwhile and upregulate Claudin-2 in vitro. A Western blotting analysis confirmed exosomes isolated from GES-1 and CagA H. pylori coculture media via characteristic biomarkers HSP70 and CD9. The presence of CagA within the exosome was also shown. Exo-CM, exosome derived from conditioned medium. B, C Features of exosomes in terms of size distribution (B) and morphology (C) on transmission electron microscopy. Scale + + bar, 0.5 µm and 200 nm. D Western blotting verified the augmentation of Claudin-2 proteins by CagA exosomes, while the CagA exosome group partially maintained Claudin-2 protein expression under IFN-γ conditions. p-STAT1 and GBP1 were employed as positive controls to + − show the inflammatory status in response to CagA exosomes, CagA exosomes, or IFN-γ. The p-STAT1 pathway was stringently dependent on IFN-γ, as expected, and the presence of a slight GBP1 band also showed a low degree of inflammation after the entry of CagA exosomes. The shrinkage of ZO-1, an acknowledged tight junction protein in both CagA exosomes and IFN-γ conditions, was observed as a positive control. E Cell immunostaining showed that CagA exosomes contributed to membrane Claudin-2 formation, whose increase was also observed in IFN-γ induced barrier function disorders. Scale bar, 20 µm. F RT–qPCR revealed the mRNA expression tendencies in different groups. G CagA -containing exosomes accelerate the dysintegrity of the NCM460 cell monolayer under inflammatory factor IFN-γ conditions. The transepithelial electrical resistance ( TEER) values of the cell monolayer were tested after CagA exosomes and IFN-γ 24 h. The data are expressed as the means ± SD of three independent experiments. ***p < 0.001, t test Guo  et al. Gut Pathogens (2022) 14:13 Page 5 of 13 Fig. 2 (See legend on previous page.) challenge with IFN-γ in NCM460 monolayers. IFN-γ expression of phospho-STAT1 and guanylate-binding treatment triggered Claudin-2 upregulation and dis- protein 1 (GBP1) was concurrently examined by west- rupted ZO-1 formation, as expected, while the shrink- ern blotting to confirm this specific and overall inflam - + + ing ZO-1 bands were detected both in the C agA matory response after IFN-γ and CagA exosome exosome and CagA exosome groups. The protein treatments (Fig. 2D and E). Guo et al. Gut Pathogens (2022) 14:13 Page 6 of 13 We next confirmed the increased mRNA level of Clau - activated by the CagA protein and is essential for tran- din-2 after treatment with C agA exosomes, while IFN-γ scriptional upregulation of Claudin-2. had the similar effects on their transcription (Fig.  2F). These data collectively indicate that IFN-γ and CagA CDX2‑dependent Claudin‑2 activation impairs mucosal exosomes enhance Claudin-2 protein expression via tran- barrier integrity in chronic colitis in vitro scriptional upregulation patterns. In the TEER experi- We then explored the relevant role of CDX2 in the ment, treatment with GES-1-derived CagA-containing mucosal barrier with respect to TJs, such as Claudin-2. exosomes had a slight effect on the colon monolayer When transfected with GFP-tagged CDX2, western blot- under normal conditions but significantly aggravated the ting showed increased Claudin-2 expression. Treatment collapse of mucosal integrity after cytokine IFN-γ inter- with IFN-γ induced colonic barrier damage, manifested ference (Fig.  2G). Notably, both CCK-8 and EdU assays as significantly upregulated expression of Claudin-2, confirmed that CagA-containing exosomes and IFN-γ which was also detected at higher levels in CDX2-over- stimulation did not cause changes in the proliferation or expressing cells (Fig.  4A). These results were consistent confluence of NCM460 cell monolayers (Additional file  6: with the TEER value tendency affected by IFN-γ and Figure S4A and B). It is thus tempting to speculate that CDX2 (Fig.  4B). In further experiments, polarized mon- the alteration of Claudin-2 by CagA-containing exosomes olayers of NCM460 vector control and CDX2 stable over- and IFN-γ negatively correlates with the integrity of the expressing cells were wounded and then incubated with intestinal mucosal barrier. IFN-γ. In untreated control and CDX2-overexpressing cells, approximately 89.1% and 87.2% of the wounded areas spontaneously healed at 24 h after wounding, indi- CagA‑associated Claudin‑2 transcriptional upregulation cating that CDX2 treatment slightly reduced migration is dependent on CDX2 activation but had no significant difference between the two groups. Based on the above results showing that Claudin-2 In control cells treated with IFN-γ, the healing rate was expression is transcriptionally regulated, we attempted to inhibited even further to 70.1%, whereas in CDX2-over- investigate whether potential transcription factors could expressing monolayers treated with IFN-γ, 45.7% of the act as decisive intermediates between exosome-carried wounded area was healed (Fig.  4C). These data indicate CagA proteins and downstream TJs. The transcription that CDX2 positively regulates Claudin-2 expression and factor CDX2 (Caudal related homeodomain transcrip- impairs mucosal barrier and wound healing functions in tion 2) was predicted to regulate Claudin-2 expression chronic colitis conditions in vitro. according to the Jasper database (Fig. 3A). In addition to exosomes from C agA H. pylori and GES-1 cell cocul- Discussion tures, human recombinant His-CagA protein was added Abundant evidence indicates that chronic H. pylori infec- to GES-1 cells, followed by exosome isolation. Signifi - tion may contribute to the development of IBD. There - cantly activated CDX2 was detected in exosomes from fore, it is essential to identify the mechanisms mediating GES-1 cells treated with both CagA H. pylori and a disease initiation and progression for the development foreign recombinant CagA protein, and the western blot of prevention and treatment strategies for IBD. H. pylori results indicated that CagA, the main virulence factor of mostly resides in the gastric epithelial layer, and a frac- H. pylori, played a prominent role in CagA-containing tion of H. pylori can be found in the lamina propria of exosome-mediated mechanisms (Fig. 3B). the submucosa close to gastric mucosa capillaries and The ChIP results showed that CDX2 could pull down postcapillary venules [38, 39]. However, no H. pylori has Claudin-2 DNA fragments, and CDX2 overexpression/ been detected in the blood of infected patients to directly depletion resulted in significantly increased/decreased affect colitis, and there is no evidence to define a causal Claudin-2 signals (Fig. 3C). A luciferase reporter assay in relationship between this “remote infection” and intesti- a HEK293T cell model confirmed the binding of CDX2 in nal mucosal integrity [40]. An exosome is a cell-derived Claudin-2 promoters and a positive transcriptional pat- vesicle that also plays an important role in cell-to-cell tern (Fig.  3D). Furthermore, CDX2 knockdown exhib- communication. The regulation of exosome-associated ited significantly greater inhibitory effects on Claudin-2 IBD has attracted increasing attention due to its poten- transcription and translation, even under conditions of tial exosome pathway function. Exosome-carried CagA CagA-containing exosome exposure, suggesting that protein can enter the circulation and transfer microbial Claudin-2 was mainly driven by CDX2 (Fig.  3E and F). virulence factors to sites far from the primary disease, Coimmunoprecipitation further precluded the protein which may cause seemingly unrelated clinical effects or interaction of nucleoprotein CDX2 and membrane pro- symptoms [36]. This was also confirmed in our study, in tein Claudin-2 (Fig.  3G). Collectively, CDX2 could be which exosomal CagA was detected in the serum of mice Guo  et al. Gut Pathogens (2022) 14:13 Page 7 of 13 Fig. 3 CagA activates CDX2 to transcriptionally upregulate Claudin-2 expression. A Schematic binding prediction of the transcription factor CDX2 in the Claudin-2 gene promoter. The two positions and sequences with relatively high scores are indicated. B Western blotting confirmed the incremental CDX2 protein expression by CagA. In addition to exosomes from GES-1 gastric epithelial cells cultured with C agA H. pylori, recombinant His-CagA protein was utilized to perform CagA and GES-1 cell cocluture and subsequent exosome isolation. Both exosomes from CagA H. pylori infection and recombinant CagA protein incubation triggered CDX2 expression. The presence of CagA in exosomes was proven using an antibody against the His-tag. Notably, the more striking contrast of the CDX2 band was shown above at shorter exposure times due to its abundant endogenous expression in the colon. C Identification of the CDX2-Claudin-2 interaction using chromatin immunoprecipitation. CDX2 interferences were introduced into NCM460 cell. CDX2 protein was able to pull down Claudin-2 DNA fragments. IgG and Input were performed as controls. D The positive transcriptional regulation of Claudin-2 by CDX2 was confirmed by luciferase reporter assay. The ratio of firefly luciferase activity to Renilla activity was calculated to show the binding of CDX2 to gene promoter activities. Each experiment was performed in triplicate. E, F CDX2 depletion impeded CagA-associated Claudin-2 transcription and protein expression. Data are presented as the mean ± SD value from three biological replicates. **p < 0.01, ***p < 0.001, t test. G Coimmunoprecipitation precluded the protein interaction of CDX2 and Claudin-2. The equal CDX2 loading in the IP panel was preverified, and immunoblotting with Claudin-2 provided no binding of CDX2 and Claudin-2 even under the longest exposure H. pylori, such as retarded growth and unsuccessful bac- infected with CagA H. pylori. We demonstrated that terial infection, reduced bacterial adhesion and inter- exosomal CagA from H. pylori disrupts the barrier func- nalization in cell line model [41]. Even, in the multiple tion of intestinal epithelial cells in colitis by facilitating knockout mutants, CagA and VacA, the key virulence Claudin-2 expression. factors of H. pylori, were both substantially reduced Ideally, a cagA knockout strain derived from the cor- [42]. Future studies are needed to explore whether there responding CagA strain is better used for the control. are significant differences among CagA wild-type, CagA However, we currently have some concerns on using the depleted, and CagA mutated H. pylori strains in vivo and knockout mutants, since recent publications shown that in vitro. knocking out gene affects the structure and function of Guo et al. Gut Pathogens (2022) 14:13 Page 8 of 13 Fig. 4 CDX2 is indispensable for Claudin-2 expression in mucosal barrier integrity and colonic epithelial restitution. A Overexpression of CDX2 upregulated Claudin-2 protein expression, and their abundances were detected even under IFN-γ incubation. The stable CDX2 overexpression model was employed using GFP-tagged vectors. B CDX2 partially impaired colonic mucosal barrier integrity in IFN-γ stimulation. The TEER values of the NCM460 cell monolayer under each condition were measured. **p < 0.01, ***p < 0.001, t test. C CDX2-associated Claudin-2 impeded colonic wound healing and partially strengthened the IFN-γ-induced mucosal deficiency. The wounds were recorded at 0 h, 6 h, 12 h, and ultimate 24 h. CDX2 + IFN-γ vs. IFN-γ, ***p < 0.001, t test CDX2 is a transcription factor that has been shown with a more metastatic phenotype of gastric carcinoma to activate the Claudin-2 promoter in human intesti- cells [49], whereas CDX2 might act as a tumor suppres- nal epithelial cells and plays a crucial role in controlling sor during colorectal carcinogenesis [50]. These discrep - the balance between proliferation and differentiation of ancies led us to explore the fine regulation of CDX2 in intestinal epithelial cells [43–45]. An indirect pattern of both gastric and intestine epithelial cells as the C agA ZO-1 regulation by CDX2 via the MEK/ERK pathway H. pylori related primary lesion and secondary effect, was also verified in porcine intestinal epithelium [46]. respectively. CDX2 expression is significantly decreased in patients In summary, we demonstrated for the first time that with active UC, and it is involved in protection against chronic infection with CagA H. pylori exacerbates DSS-induced colitis [47, 48]. However, to date, the regu- DSS-induced UC-like colitis in mice. A series of in  vitro lation of mucosal inflammation by CDX2 as a triggering experiments confirmed the exosomal CagA-associated or compensative factor is rather unclear. Interestingly, a upregulation of Claudin-2 expression by the activation recent study indicated that CagA disrupts tight junctions of the transcription factor CDX2. The damaging role of by increasing the CDX2-dependent targeting of Clau- CagA in mucosal barrier integrity was verified by a series din-2 in gastric carcinoma cells and causes significant of functional experiments under IFN-γ-stimulating con- changes in the morphology and activity of these cells ditions. Although it is not clear whether intestinal epi- [49]. In our experiment, exosomal CagA upregulated thelial barrier dysfunction is a cause or a consequence of CDX2 expression and increased Claudin-2 expression to IBD, intestinal epithelial Claudin-2 can still be used as a impair the intestinal mucosal barrier. Similarities in these target to impair the integrity of the IBD intestinal barrier, results may be due to the infiltration of CagA exosomes which provides a new idea for IBD treatment. Further into either the gastric or colonic epithelium irrespective research is needed to determine whether such a mecha- of the different and complex intracellular environments. nism may also be involved in other extragastric diseases Moreover, increased CDX2 expression was associated caused by H. pylori infection. Guo  et al. Gut Pathogens (2022) 14:13 Page 9 of 13 Materials and methods without CagA H. pylori at a multiplicity of infection H. pylori culture of 100 for 2, 6, and 12  h. The conditioned media were + − CagA H. pylori and CagA H. pylori used in culture then collected from the coculture systems to isolate the and mouse studies were isolated from gastric specimens exosomes as described [37]. Briefly, cells and debris in of gastric ulcer patients during gastroscopy as described conditioned media were eliminated by successive cen- [37, 51]. In brief, bacteria were grown on Columbia blood trifugation (4 °C) at increasing speeds (300 g for 10 min, agar plates supplemented with antibiotics (10 mg/L van- 2000 g for 20 min, then 10,000 g for 30 min). The super - comycin, 5  mg/L cefsulodin, 5  mg/L amphotericin B, natant was next transferred to an ultracentrifuge tube 5  mg/L trimethoprim, and 10% sheep blood (Thermo and centrifuged at 100,000  g at 4  °C for 70  min twice Scientific R54008, Waltham, MA)) at 37 °C under micro - (Beckman Coulter, Indianapolis, IN). Exosome pellets aerophilic conditions (5% O , 10% CO , and 85% N ) for were resuspended in a small volume containing PBS for 2 2 2 3–4  days. After incubation for 3–4  days, the bacterial further analysis. The protein level in the exosomes was colonies were collected by scrape and then resuspended determined by the BCA protein assay (Cat.No.23235; in PBS. The bacterial concentration was calculated based Thermo Fisher, Waltham, MA). The morphologies, size on an optical density (OD) of 1 × 10 colony-forming distribution, and biomarkers (HSP70 and CD9) of the units (CFUs) at 660 nm. Identification of CagA H. pylori exosomes were examined using a transmission electron was confirmed using the complete sequence data of the microscopy (TECNAI G2 Spirit; FEI, Hillsboro, OR), H. pylori 16S rRNA gene from GenBank data (sequence dynamic light scattering with a particle and molecular ID AP017362) and positive biochemical test reactions for size analyzer (Zetasizer Nano ZS; Malvern Panalytical, oxidase, catalase, and urease. Malvern, Worcestershire, UK), and western blotting, respectively. Cell culture and cell‑bacteria coculture The human gastric epithelial cell line GES-1 and intes - Cell viability and migration assay tinal epithelial cell line NCM460 were continuously Cell proliferation was analyzed using a commercial cultured in Dulbecco’s modified Eagle’s medium (Sigma– CCK-8 assay kit (#C0038, Beyotime) and EdU detection Aldrich, USA) containing 10% fetal bovine serum (FBS) kits (RiboBio, Guangzhou, China). The wound scratch (Gibco, New York, USA), 100  IU/mL penicillin G and assay was performed to evaluate the migration ability. 100 μg/mL streptomycin (Sigma–Aldrich, USA) in a con- Polarized monolayers of NCM460 cells on transparent trolled humidified incubator at 37 °C with 5% CO . supports were washed twice with prewarmed PBS, and wounds were created by scraping the monolayers with a Reagents, plasmid construction, RNA interference 10  nm pipette tip. Media containing drugs (IFN-γ) was and transfection then applied. Twelve random wound points were chosen, The chemical reagents IFN-γ (cat. #PHC4033) was from and images were taken at time 0  h, 6  h, 12  h, and 24  h. Gibco (Houston, TX, USA). The recombinant His-CagA The wound closure area was measured using NIH ImageJ protein and the full-length CDX2 plasmid tagged with software. GFP were purchased from GeneChem Biotechnol- ogy Company (Shanghai, China). Short hairpin RNA (shRNA) for CDX2 and its corresponding negative TEER measurement control were also synthesized by GeneChem (Shang- To evaluate the barrier integrity of NCM460 cells, the hai, China). Transfections were conducted using Lipo- transepithelial electrical resistance (TEER) was meas- fectamine 2000 (Invitrogen) according to a previously ured. The initial TEER was tested before the cells were described protocol [52]. Knockdown efficiencies were seeded. Colon cells with and without treatment were determined by immunofluorescence and western blot - seeded in a Transwell membrane insert (12 mm diameter, ting, and RNAi sequence with relatively high results was 0.4  μm pore size, Corning) at a density of 7 × 10   cells/ selected for further experiment. The primer informa - well. Then, 200 μL and 500 μL medium was added to the tion of CDX2, ZO-1, and Claudin-2, as well as target- apical and basal compartments, respectively. Cecropin A ing sequences of CDX2, are listed in Additional file  1: (12.5 μg/mL) was added to the apical and basal compart- Table S1. ments. The TER values were measured every day by using an ohm-meter fitted with chopstick electrodes (Millipore ESR-2; Burlington, MA, USA). Before each test, the plates Exosome isolation and identification were placed at room temperature for 30  min. The TER To prepare exosomes from conditioned media, GES-1 was calculated by using the following equation: TEER (Ω  human gastric epithelium cells were cultured with or cm ) = (TEER − TER ) × 0.3. initial Guo et al. Gut Pathogens (2022) 14:13 Page 10 of 13 Luciferase assay 2) mice administrated H. pylori via gavage only (Hp, Cells were seeded into 96-well plates and grown to n = 10); 3) mice receiving PBS via gavage and DSS (DSS, approximately 80% confluency on the second day. Next, n = 10); and 4) mice receiving CagA H. pylori via gav- the relevant reporter plasmids and the PRL-TK reporter age and DSS (Hp + DSS, n = 10). were transiently cotransfected into the cells. After 48  h, The Hp-infected groups were administered 2.5 × 10 firefly luciferase activity and Renilla activity were deter - CFU CagA H. pylori by oral gavage once every other day mined. The ratio of firefly luciferase activity to Renilla seven times over 13 days to ensure a 100% infection rate. activity was regarded as indicating the activity of the gene At the same time, the mice in the corresponding control promoters. Each experiment was performed in triplicate. group received PBS by oral gavage as the control. All mice were fasted 12  h before and 4  h after each inocu- lation. CagA H. pylori colonization in the gastric epi- Immunofluorescence staining thelium was confirmed histopathologically using gastric Cells were grown on polyethyleneimine-coated cover- + tissues from the mice after 8  weeks of CagA H. pylori slips, washed with prewarmed phosphate-buffered saline infection with Giemsa and Warthin-Starry silver staining. (PBS), fixed in 4% paraformaldehyde for 15 min, permea - + For the colitis experiments after 8  weeks of CagA bilized with 0.5% Triton X-100 in PBS for 10 min, blocked H. pylori infection, the DSS and Hp + DSS groups were with 3% bovine serum albumin (BSA) solution for 1  h, subjected to three repeated cycles of 2% DSS (Sigma, St and incubated with anti-primary antibodies in 3% BSA at Louis, MO, USA) to induce chronic colitis (7 days/cycle) 4 °C overnight (antibody information listed in Additional as instructed [53], and each was separated by 7  days of file  2: Table  S2). Cells were then rinsed three times for regular water. During the process, the body weight, dis- 5  min with PBS and incubated with individual primary ease activity index (DAI) and general status of all mice antibodies in 3% BSA at 37  °C for 90  min. Alexa488 or were observed every three days. Specifically, DAI scoring Alexa555 anti-rabbit secondary antibodies (1:100, Inv- was measured as described previously (DAI = (Weight itrogen) were added for 1  h in the dark. Nuclei were loss score + Stool characters score + Hematochezia counterstained with DAPI (1:1000, sc-3598; Santa Cruz) score)/3) [54]. Mice were sacrificed on the seventh day in PBS at room temperature for 2  min, rinsed with PBS after returning to drinking water freely. Colon length three times for 3 min, and mounted with SlowFade Gold and the weight of spleen were measured. Colon tissues Antifade Reagent (S36942, Life Technologies). Images for histological analyses were dissected and immediately were obtained from a confocal microscope (TCSSP8, fixed with 4% paraformaldehyde. Leica Microsystems) equipped with an acousto-optic The colons were opened longitudinally and briefly beam splitter, a 405-nm laser (for DAPI), an argon laser cleansed with cold PBS. Tissues were then divided into (488 nm for Alexa 488), and a diode-pumped solid-state two equal sections. The proximal parts were fixed in (DPSS) laser (561 nm). 10% formalin for histological examination while the dis- tal parts were stored at -80  °C for protein extraction. Paraffin-embedded sections of the indicated colons were Quantitative real‑time RT–PCR and RNA array subjected to H&E staining and then examined by light Quantitative real-time RT–PCR (qRT–PCR) analyses microscopy. The histological scores were analyzed as were conventionally carried out. The primers used for the described previously [55]. qPCR of the referenced genes are shown in Additional file 1: Table S1. Protein extraction, immunoprecipitation, and western blot analysis Chronic colitis models of  CagA H. pylori‑infected mice Protein extracts were obtained from fresh cells and colon All animal experiments were performed in accord- tissues with PBS and in RIPA buffer (Thermo Scientific, ance with the ARRIVE Guidelines. The study was MA, USA) supplemented with protease inhibitor and conducted according to the guidelines of the Declara- phosphatase inhibitor (Roche, Welwyn Garden, Switzer- tion of Helsinki, and approved by the Experimental land). Supernatants were eluted in SDS sample buffer and Animal Ethics Committee of the Third Xiangya Hos - analyzed by SDS–PAGE. For immunoblotting, primary pital of Central South University (The Research Code antibodies are indicated in Additional file  2: Table  S2. #LLSC(LA)2018-039, 16 January 2018). Specific-path - Blots were then developed with ECL western blotting ogen-free (SPF) male C57BL/6 mice (4  weeks) were reagents (Pierce Biotechnology, Rockford, IL). Signal cultured in SPF conditions. Forty mice were randomly intensity was quantified with ImageJ (National Institutes divided into four groups: 1) mice receiving a standard of Health, Bethesda, MD). diet and normal drinking water (control, NC, n = 10); Guo  et al. Gut Pathogens (2022) 14:13 Page 11 of 13 Chromatin immunoprecipitation (ChIP) assay Additional file 6: Figure S4. Cell proliferation and viability assay using For ChIP, 1.5 × 10 cells were subjected to a two-step dual EdU and CCK-8. These results indicated the confluence of the NCM460 cell monolayer at the beginning, and the proliferation and survival of colonic cross-linking procedure based on previously described cells were not affected by either CagA exosomes or IFN-γ stimulation. methods [56]. In brief, protein-DNA complexes were immunoprecipitated with anti-CDX2 antibodies followed Acknowledgements by q-PCR using a BioRad CFX-96 quantitative thermo- The authors are grateful to the Endoscopic Unit of Third Xiangya Hospital of cycler and SsoFast EvaGreen Low-ROX qPCR Super- Central South University for providing clinic samples to isolate H. pylori strains. Mix (BioRad). For qPCR quantification, the following Authors’ contributions primer pairs are listed in Additional file  1: Table S1. Data YG, CX, and XL designed the study, analyzed, and interpreted the data, were analyzed according to the ΔΔCT method (Applied and wrote the manuscript. RG, TH and XZ contributed to the data acquisi- Biosystems). tion, analysis, and interpretation. RG and XX (Xiaoran Xie) performed all the bioinformatics analyses. YG, TH, XZ, JC, HL and XX (Xiujuan Xia) carried out the experiments. CX and XX (Xiujuan Xia) provided technical expertise and sup- port. All authors read and approved the final manuscript. Bioinformatic and statistical analyses Funding Claudin-2 promoter (−  2000  bp) sequences (H. sapiens) This work was supported by the National Science Foundation of China were downloaded from UCSC (http:// genome. ucsc. edu/). (82002614), the Natural Science Foundation of Hunan Province (2021JJ40935), The specific binding of transcription factors in gene pro - the Wisdom Accumulation and Talent Cultivation Project of the Third Xiangya Hospital of Central South University (YX202103), the Fundamental Research moters was predicted by the Jasper database (http:// jaspa Funds of Central South University (1053320183950), and the National Innova- r2016. gener eg. net/). Statistical analyses were performed tive Training Program of China (201310533059). using SPSS 19.0 software (version 19.0; SPSS Inc., Chi- Availability of data and materials cago, IL). The results are presented as the mean ± S.D., Not applicable. and two-group comparisons were evaluated using Stu- dent’s t test. Declarations Ethics approval and consent to participate Abbreviations All the animals received human care. The study protocol conformed to the H. pylori: Helicobacter pylori; CagA: Cytotoxin-associated gene A; T4SS: Type IV ethical guidelines of the 1975 Declaration of Helsinki and was approved by secretion system; TJ: Tight junction; IBD: Inflammatory bowel disease; CDX2: the Experimental Animal Ethics Committee of the Third Xiangya Hospital of Caudal related homeodomain transcription 2; ZO: Zonula occludens; GBP1: Central South University ( The Research Code #LLSC(LA)2018-039, 16 January Guanylate-binding protein 1; TEER: Transepithelial electrical resistance; DSS: 2018). Dextran sulphate sodium. Consent for publication Not applicable. Supplementary Information The online version contains supplementary material available at https:// doi. Competing interests org/ 10. 1186/ s13099- 022- 00486-0. The authors declare that they have no competing interests. Author details Additional file 1: Table S1. Primers and shRNAs used in this study. Department of Gastroenterology, Third Xiangya Hospital of Central South Additional file 2: Table S2. Antibodies for western blot, immunoprecipi- University, 138 Tongzipo Road, Changsha 410013, China. Depar tment tation, and immunofluorescence in this study. of Ophthalmology, The Second Xiangya Hospital, Central South University, Changsha 410008, China. Hunan Key Laboratory of Nonresolving Inflamma- Additional file 3: Figure S1. Confirmation of H.pylori colonization in tion and Cancer, Changsha 410013, China. gastric mucosa by Giemsa and silver staining in C57BL/6 mice. Stainings in H. pylori alone group and H. pylori+DSS group indicated that DSS had no Received: 22 September 2021 Accepted: 10 March 2022 effects on H. pylori colonization. scale bar, 20µm. Additional file 4: Figure S2. 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Exosomal CagA from Helicobacter pylori aggravates intestinal epithelium barrier dysfunction in chronic colitis by facilitating Claudin-2 expression

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Abstract

Background: The chronic infection with Helicobacter pylori (H. pylori), especially cytotoxin-associated gene A-positive (CagA ) strains, has been associated with various extragastric disorders. Evaluating the potential impacts of virulence factor CagA on intestine may provide a better understanding of H. pylori pathogenesis such as colitis. The intestinal mucosal barrier is essential for maintaining its integrity and functions. However, how persistent C agA H. pylori coloni- zation influences barrier disruption and thereby affects chronic colitis is not fully understood. Results: Chronic colitis models of CagA H. pylori-colonized mice treated with 2% Dextran sulphate sodium (DSS) were established to assess the disease activity and pertinent expression of tight junction proteins closely related to mucosal integrity. The aggravating effect of CagA H. pylori infection on DSS-induced chronic colitis was confirmed in mouse models. In addition, augmented Claudin-2 expression was detected in C agA H. pylori infection conditions and selected for mechanistic analysis. Next, GES-1 human gastric epithelial cells were cultured with C agA H. pylori or a recombinant CagA protein, and exosomes isolated from conditioned media were then identified. We assessed the + − + Claudin-2 levels after exposure to CagA exosomes, CagA exosomes, and IFN-γ incubation, revealing that CagA H. pylori compromised the colonic mucosal barrier and facilitated IFN-γ-induced intestinal epithelial destruction through CagA-containing exosome-mediated mechanisms. Specifically, CagA upregulated Claudin-2 expression at the tran- scriptional level via a CDX2-dependent mechanism to slow the restoration of wounded mucosa in colitis in vitro. Conclusions: These data suggest that exosomes containing CagA facilitate CDX2-dependent Claudin-2 main- tenance. The exosome-dependent mechanisms of CagA H. pylori infection are indispensable for damaging the mucosal barrier integrity in chronic colitis, which may provide a new idea for inflammatory bowel disease (IBD) treatment. Keywords: CagA, Exosome, Colitis, Tight junction, Claudin-2, CDX2 Introduction Helicobacter pylori (H. pylori), a Gram-negative micro- aerophilic bacterium in the stomach, is one of the most ubiquitous pathogens as it colonizes more than half of the *Correspondence: xiaxiujuan2007@163.com; liuxiaoming26@163.com world’s population [1]. H. pylori infection is closely asso- Department of Gastroenterology, Third Xiangya Hospital of Central South University, 138 Tongzipo Road, Changsha 410013, China ciated with several gastric diseases, including gastritis, Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Guo et al. Gut Pathogens (2022) 14:13 Page 2 of 13 peptic ulcer disease, and gastric cancer [2]. Among the immunomodulatory effect in IBD since its DNA inhibits reported pathogenic factors of H. pylori, cytotoxin- proinflammatory cytokine production of dendritic cells, associated gene A protein (CagA) has been the focus of equilibrates Th17/Treg and Breg cell responses, and shifts attention. CagA is a specific virulence factor protein of macrophages toward M2 anti-inflammatory environ - H. pylori that enters host cells through the type IV secre- ments [27–30]. Intriguingly, when considering the exact tion system (T4SS). Once inside the cell, CagA triggers a pathogenic or protective effects of CagA on IBD or colitis variety of signal transduction pathways, induces changes at the molecular levels, the conclusions are lacking. For in cell morphology, and increases the risk of gastric dis- example, dextran sulphate sodium (DSS)-induced colitis eases [3, 4]. Moreover, increasing evidences show that was prominently deteriorated in CagA-transgenic mice + − CagA H. pylori strains other than C agA H. pylori are [31]. The biological basis for this effect regarding mucosal pathogens closely related to diseases outside the stom- barrier integrity remains unclear. ach [5, 6]. Deposited in renal tubules, CagA brings about Exosomes are extracellular vesicles derived from mul- strongly mucosal immune response in IgA nephropathy tivesicular bodies and are secreted by healthy cells, can- [7]. CagA H. pylori strains induce premature senescence cer cells, and host cells [32]. During infection, exosomes of extragastric cells, which may contribute to chronic released from host cells can convey pathogenic compo- skin diseases [8]. CagA seropositivity are highly corre- nents [33]. Exosomes are key regulators of cellular physi- lated with sarcopenia and low muscle quantity [9]. In the ological functions and pathogenesis [34]. A recent study context of CagA’s contribution to extragastric diseases, a reported that exosomes containing CagA were released large proportion of conundrums such as inflammatory from H. pylori CagA-expressing cells and could be bowel disease (IBD) remain to be fully elucidated. detected in blood circulation [35]. Moreover, exosomes Among the defensive mechanisms of the intestinal released from H. pylori-infected host gastric epithelial mucosa, an impaired epithelial barrier is the main path- cells can enter the bloodstream and aortic plaques, pro- ological factor that causes colitis, and it contributes to mote the formation of macrophage-derived foam cells, increased permeability by allowing increased antigen and accelerate the progression of atherosclerosis [36, 37]. penetration thereby initiating and propagating the dys- u Th s, exosome-carried CagA protein may be involved regulated response [10–13]. The normal intestinal bar - in the development of a variety of extragastric diseases rier consists of an intact layer of epithelial cells, which including IBD. are connected by a system of tight junction (TJ) strands To elucidate how H. pylori infection could affect IBD- [14]. TJs are composed of the occludin and claudin fam- associated chronic colitis via TJs, mouse models were ily members, which are transmembrane proteins, as well established, and in  vitro experiments were conducted in as TJ-associated proteins, such as Claudin-2 and ZO-1 the present study. CagA H. pylori infection was used to [15, 16]. Claudin-2 has been shown to induce cation- test the hypothesis that exosomal CagA from H. pylori selective channels in the tight junctions of epithelial cells, compromised the barrier integrity of the intestinal epi- and its expression is increased in IBD, leading to diar- thelium in colitis by facilitating Claudin-2 expression. rhea through the leakage flux mechanism [17, 18]. It was recently reported that Claudin-2 degradation enhances Results TJ barrier function in intestinal epithelial cells [19]. Spe- CagA H. pylori infection increases Claudin‑2 expression cifically, Claudin-2 expression is positively associated and aggravates DSS‑induced chronic colitis in C57BL/6 with inflammatory activity in patients with IBD, and plays mice multiple roles, such as targeting the vitamin D receptor, To determine the effect of H. pylori on the severity of in IBD pathogenesis [20, 21]. Another TJ protein, ZO-1, DSS-induced colitis, we first established a H. pylori- connects occludins and claudins to cytoskeletal actin, infected C57BL/6 mouse model under chronic inflam - and the loss of ZO-1 leads to increased intestinal per- mation conditions (Fig.  1A). Histopathological analysis meability [16]. ZO-1 preserves the intestinal barrier, and of the mouse gastric tissues of the mice after 8  weeks decreased intestinal inflammation has been observed [22, of H. pylori (CagA strain) infection showed positive 23]. To the best of our knowledge, the potential impli- Giemsa and silver staining for H. pylori in all the tested cations of TJ dysregulation in IBD pathogenesis remain animals, confirming that the mouse model of H. pylori elusive. infection was successful (Additional file  3: Figure S1). Certain clinical studies and systematic reviews have We then compared and analyzed the body weight and indicated the association between H. pylori exposure disease activity index (DAI) of mice with and without H. and reduced IBD risk [24–26]. The clinically observed pylori infection every three days after 2% DSS adminis- inverse association between H. pylori and IBD could be tration. The anatomical and histopathological features of partly explained by the fact that H. pylori might exert an the colon and spleen in each group were also evaluated. Guo  et al. Gut Pathogens (2022) 14:13 Page 3 of 13 Fig. 1 CagA H. pylori infection exacerbates DSS-induced chronic colitis in mice. A Schematic depiction of the experimental design for H. pylori (CagA strain) inoculation followed by chronic colitis models administrated 2% DSS. B, C Body weights (B) and disease activity index (DAI) changes (C) in different groups. Data from the second and third DSS-treatment cycles (7 day 2% DSS and 7 day diluted water) showed that H. pylori-infected mice had significantly less body weight loss and disease manifestation than control mice with no H. pylori infection in DSS-treated conditions. ***p < 0.001, compared to DSS group. Student’s t test for body weights, and χ test for DAI scores. D, E, F Colon lengths (D), spleen weights (E), microscopic appearance and H&E histological sections of the colon F in each group. Scale bars, 200 µm (200 ×) and 100 µm (400 ×). **p < 0.01, ***p < 0.001. Student’s t test was used for colon length and spleen weights, and the χ test was used for histological scores. G The relative expression of tight junction proteins ( TJs) in the colon of H. pylori and chronic colitis mice. The augmented Claudin-2 was observed in the H. pylori + DSS group in comparison with the DSS group. H The TJs protein expression of chronic colitis mice subgroups without H. pylori, with C agA H. pylori infection, + + − and with CagA H. pylori infection. Claudin-2 was significantly upregulated in CagA H. pylori-infected group compared with CagA H. pylori mice. All data were presented as means ± SD (n = 10) Guo et al. Gut Pathogens (2022) 14:13 Page 4 of 13 As expected, the DSS group showed significant weight colitis conditions (CagA negative status was identi- loss and higher DAI scores. In comparison with the nor- fied in Additional file  4: Figure S2). The colon histologi - mal control (NC) group, the mice with H. pylori infec- cal sections, colon lengths, and spleen weight verified tion alone (Hp group) displayed no significant changes that aggravated inflammation and intestinal injury were + − in weight or DAI scores. However, the mice pretreated detected in CagA H.pylori group compared to C agA with H. pylori infection exhibited more weight loss and H.pylori group (Additional file  5: Figure S3A, B, and manifestations than the DSS-treated group, and the more C). As for TJs tendency, claudin-2 levels in CagA H. intriguing finding was that these damaging effects of H. pylori group was markedly increased in comparison with − − pylori colonization on chronic colitis were obviously CagA H.pylori group. Intriguingly, CagA H.pylori was detected in the second and third 2% DSS cycles (Fig.  1B, not responsible for upregulated claudin-2 compared to C). chronic colitis mice subgroup without H. pylori infec- All the mice with DSS-induced colitis showed vary- tion (Fig.  1H). Collectively, CagA H. pylori infection ing degrees of shorter intestine, larger spleen weight, increases Claudin-2 expression and aggravates DSS- and loss of crypt and goblet cells with distinct mucosal induced chronic colitis. and submucosal infiltration of inflammatory cells, while H. pylori-pretreated mice showed significantly distinct CagA‑containing exosomes upregulate Claudin‑2 anatomical and histopathological abnormities (Fig.  1D, expression to compromise the intestinal mucosal barrier E, and F). The above data suggest a possible injuring role integrity in vitro of H. pylori in enhancing DSS-induced chronic colitis, To determine how CagA H. pylori infection could affect which is associated with incomplete regulation of the DSS-induced colitis, we hypothesized that CagA H. mucosal epithelium by TJs. Western blot analysis of the pylori exerted its function through CagA-containing expression of potential TJs was performed in the control exosome-mediated mechanisms. Typical exosomes were and experimental groups. Claudin-2 level was gradu- present in the conditioned media of GES-1 cells cul- ally increased in Hp and DSS group, reaching the peak tured with CagA H. pylori and were characterized by in Hp + DSS groups, as expected, while claudin-2 levels their specific biomarkers (HSP70, CD9, and CagA), size gradually increased in the Hp and DSS groups, peaking in distribution, and morphology (Fig.  2A, B, and C). These the Hp + DSS group. ZO-1 expression was substantially western blotting and electron microscopy results con- downregulated in the DSS groups, as expected, while the firm the uptake of exosomes by NCM460 cells, which is increasing tendency of ZO-1 in Hp + DSS group is not analogous to our previous immunofluorescence staining obvious. No changes in occludin were observed among results that the unique H. pylori pathogenic factor CagA the abovementioned groups (Fig. 1G). entered human GES-1 cells after they were incubated The trend of Claudin-2 expression might explain how with CagA H. pylori [37]. H. pylori exerts its aggravating effect in DSS-induced Compared to C agA H. pylori strain, western blot chronic colitis. Since the utilized bacterial in previ- and immunofluorescence showed that NCM460 ous study was C agA H.pylori strain, we tried our best cells exposed to exosomes from CagA H. pylori and to address if CagA, the strongest virulent factor for H. GES-1 coculture conditions directly augmented Clau- pylori, was involved in varied barrier function in chronic din-2 protein expression while depleting ZO-1 lev- + − colitis status. Both C agA H.pylori and C agA H.pylori els. Mucosal integrity and inflammatory condition were inoculated into mice followed by 2% DSS induced were characterized before and after an inflammatory (See figure on next page.) Fig. 2 Exosomes from GES-1 human gastric epithelial cells cultured with CagA H. pylori significantly disrupt the intestinal mucosal barrier meanwhile and upregulate Claudin-2 in vitro. A Western blotting analysis confirmed exosomes isolated from GES-1 and CagA H. pylori coculture media via characteristic biomarkers HSP70 and CD9. The presence of CagA within the exosome was also shown. Exo-CM, exosome derived from conditioned medium. B, C Features of exosomes in terms of size distribution (B) and morphology (C) on transmission electron microscopy. Scale + + bar, 0.5 µm and 200 nm. D Western blotting verified the augmentation of Claudin-2 proteins by CagA exosomes, while the CagA exosome group partially maintained Claudin-2 protein expression under IFN-γ conditions. p-STAT1 and GBP1 were employed as positive controls to + − show the inflammatory status in response to CagA exosomes, CagA exosomes, or IFN-γ. The p-STAT1 pathway was stringently dependent on IFN-γ, as expected, and the presence of a slight GBP1 band also showed a low degree of inflammation after the entry of CagA exosomes. The shrinkage of ZO-1, an acknowledged tight junction protein in both CagA exosomes and IFN-γ conditions, was observed as a positive control. E Cell immunostaining showed that CagA exosomes contributed to membrane Claudin-2 formation, whose increase was also observed in IFN-γ induced barrier function disorders. Scale bar, 20 µm. F RT–qPCR revealed the mRNA expression tendencies in different groups. G CagA -containing exosomes accelerate the dysintegrity of the NCM460 cell monolayer under inflammatory factor IFN-γ conditions. The transepithelial electrical resistance ( TEER) values of the cell monolayer were tested after CagA exosomes and IFN-γ 24 h. The data are expressed as the means ± SD of three independent experiments. ***p < 0.001, t test Guo  et al. Gut Pathogens (2022) 14:13 Page 5 of 13 Fig. 2 (See legend on previous page.) challenge with IFN-γ in NCM460 monolayers. IFN-γ expression of phospho-STAT1 and guanylate-binding treatment triggered Claudin-2 upregulation and dis- protein 1 (GBP1) was concurrently examined by west- rupted ZO-1 formation, as expected, while the shrink- ern blotting to confirm this specific and overall inflam - + + ing ZO-1 bands were detected both in the C agA matory response after IFN-γ and CagA exosome exosome and CagA exosome groups. The protein treatments (Fig. 2D and E). Guo et al. Gut Pathogens (2022) 14:13 Page 6 of 13 We next confirmed the increased mRNA level of Clau - activated by the CagA protein and is essential for tran- din-2 after treatment with C agA exosomes, while IFN-γ scriptional upregulation of Claudin-2. had the similar effects on their transcription (Fig.  2F). These data collectively indicate that IFN-γ and CagA CDX2‑dependent Claudin‑2 activation impairs mucosal exosomes enhance Claudin-2 protein expression via tran- barrier integrity in chronic colitis in vitro scriptional upregulation patterns. In the TEER experi- We then explored the relevant role of CDX2 in the ment, treatment with GES-1-derived CagA-containing mucosal barrier with respect to TJs, such as Claudin-2. exosomes had a slight effect on the colon monolayer When transfected with GFP-tagged CDX2, western blot- under normal conditions but significantly aggravated the ting showed increased Claudin-2 expression. Treatment collapse of mucosal integrity after cytokine IFN-γ inter- with IFN-γ induced colonic barrier damage, manifested ference (Fig.  2G). Notably, both CCK-8 and EdU assays as significantly upregulated expression of Claudin-2, confirmed that CagA-containing exosomes and IFN-γ which was also detected at higher levels in CDX2-over- stimulation did not cause changes in the proliferation or expressing cells (Fig.  4A). These results were consistent confluence of NCM460 cell monolayers (Additional file  6: with the TEER value tendency affected by IFN-γ and Figure S4A and B). It is thus tempting to speculate that CDX2 (Fig.  4B). In further experiments, polarized mon- the alteration of Claudin-2 by CagA-containing exosomes olayers of NCM460 vector control and CDX2 stable over- and IFN-γ negatively correlates with the integrity of the expressing cells were wounded and then incubated with intestinal mucosal barrier. IFN-γ. In untreated control and CDX2-overexpressing cells, approximately 89.1% and 87.2% of the wounded areas spontaneously healed at 24 h after wounding, indi- CagA‑associated Claudin‑2 transcriptional upregulation cating that CDX2 treatment slightly reduced migration is dependent on CDX2 activation but had no significant difference between the two groups. Based on the above results showing that Claudin-2 In control cells treated with IFN-γ, the healing rate was expression is transcriptionally regulated, we attempted to inhibited even further to 70.1%, whereas in CDX2-over- investigate whether potential transcription factors could expressing monolayers treated with IFN-γ, 45.7% of the act as decisive intermediates between exosome-carried wounded area was healed (Fig.  4C). These data indicate CagA proteins and downstream TJs. The transcription that CDX2 positively regulates Claudin-2 expression and factor CDX2 (Caudal related homeodomain transcrip- impairs mucosal barrier and wound healing functions in tion 2) was predicted to regulate Claudin-2 expression chronic colitis conditions in vitro. according to the Jasper database (Fig. 3A). In addition to exosomes from C agA H. pylori and GES-1 cell cocul- Discussion tures, human recombinant His-CagA protein was added Abundant evidence indicates that chronic H. pylori infec- to GES-1 cells, followed by exosome isolation. Signifi - tion may contribute to the development of IBD. There - cantly activated CDX2 was detected in exosomes from fore, it is essential to identify the mechanisms mediating GES-1 cells treated with both CagA H. pylori and a disease initiation and progression for the development foreign recombinant CagA protein, and the western blot of prevention and treatment strategies for IBD. H. pylori results indicated that CagA, the main virulence factor of mostly resides in the gastric epithelial layer, and a frac- H. pylori, played a prominent role in CagA-containing tion of H. pylori can be found in the lamina propria of exosome-mediated mechanisms (Fig. 3B). the submucosa close to gastric mucosa capillaries and The ChIP results showed that CDX2 could pull down postcapillary venules [38, 39]. However, no H. pylori has Claudin-2 DNA fragments, and CDX2 overexpression/ been detected in the blood of infected patients to directly depletion resulted in significantly increased/decreased affect colitis, and there is no evidence to define a causal Claudin-2 signals (Fig. 3C). A luciferase reporter assay in relationship between this “remote infection” and intesti- a HEK293T cell model confirmed the binding of CDX2 in nal mucosal integrity [40]. An exosome is a cell-derived Claudin-2 promoters and a positive transcriptional pat- vesicle that also plays an important role in cell-to-cell tern (Fig.  3D). Furthermore, CDX2 knockdown exhib- communication. The regulation of exosome-associated ited significantly greater inhibitory effects on Claudin-2 IBD has attracted increasing attention due to its poten- transcription and translation, even under conditions of tial exosome pathway function. Exosome-carried CagA CagA-containing exosome exposure, suggesting that protein can enter the circulation and transfer microbial Claudin-2 was mainly driven by CDX2 (Fig.  3E and F). virulence factors to sites far from the primary disease, Coimmunoprecipitation further precluded the protein which may cause seemingly unrelated clinical effects or interaction of nucleoprotein CDX2 and membrane pro- symptoms [36]. This was also confirmed in our study, in tein Claudin-2 (Fig.  3G). Collectively, CDX2 could be which exosomal CagA was detected in the serum of mice Guo  et al. Gut Pathogens (2022) 14:13 Page 7 of 13 Fig. 3 CagA activates CDX2 to transcriptionally upregulate Claudin-2 expression. A Schematic binding prediction of the transcription factor CDX2 in the Claudin-2 gene promoter. The two positions and sequences with relatively high scores are indicated. B Western blotting confirmed the incremental CDX2 protein expression by CagA. In addition to exosomes from GES-1 gastric epithelial cells cultured with C agA H. pylori, recombinant His-CagA protein was utilized to perform CagA and GES-1 cell cocluture and subsequent exosome isolation. Both exosomes from CagA H. pylori infection and recombinant CagA protein incubation triggered CDX2 expression. The presence of CagA in exosomes was proven using an antibody against the His-tag. Notably, the more striking contrast of the CDX2 band was shown above at shorter exposure times due to its abundant endogenous expression in the colon. C Identification of the CDX2-Claudin-2 interaction using chromatin immunoprecipitation. CDX2 interferences were introduced into NCM460 cell. CDX2 protein was able to pull down Claudin-2 DNA fragments. IgG and Input were performed as controls. D The positive transcriptional regulation of Claudin-2 by CDX2 was confirmed by luciferase reporter assay. The ratio of firefly luciferase activity to Renilla activity was calculated to show the binding of CDX2 to gene promoter activities. Each experiment was performed in triplicate. E, F CDX2 depletion impeded CagA-associated Claudin-2 transcription and protein expression. Data are presented as the mean ± SD value from three biological replicates. **p < 0.01, ***p < 0.001, t test. G Coimmunoprecipitation precluded the protein interaction of CDX2 and Claudin-2. The equal CDX2 loading in the IP panel was preverified, and immunoblotting with Claudin-2 provided no binding of CDX2 and Claudin-2 even under the longest exposure H. pylori, such as retarded growth and unsuccessful bac- infected with CagA H. pylori. We demonstrated that terial infection, reduced bacterial adhesion and inter- exosomal CagA from H. pylori disrupts the barrier func- nalization in cell line model [41]. Even, in the multiple tion of intestinal epithelial cells in colitis by facilitating knockout mutants, CagA and VacA, the key virulence Claudin-2 expression. factors of H. pylori, were both substantially reduced Ideally, a cagA knockout strain derived from the cor- [42]. Future studies are needed to explore whether there responding CagA strain is better used for the control. are significant differences among CagA wild-type, CagA However, we currently have some concerns on using the depleted, and CagA mutated H. pylori strains in vivo and knockout mutants, since recent publications shown that in vitro. knocking out gene affects the structure and function of Guo et al. Gut Pathogens (2022) 14:13 Page 8 of 13 Fig. 4 CDX2 is indispensable for Claudin-2 expression in mucosal barrier integrity and colonic epithelial restitution. A Overexpression of CDX2 upregulated Claudin-2 protein expression, and their abundances were detected even under IFN-γ incubation. The stable CDX2 overexpression model was employed using GFP-tagged vectors. B CDX2 partially impaired colonic mucosal barrier integrity in IFN-γ stimulation. The TEER values of the NCM460 cell monolayer under each condition were measured. **p < 0.01, ***p < 0.001, t test. C CDX2-associated Claudin-2 impeded colonic wound healing and partially strengthened the IFN-γ-induced mucosal deficiency. The wounds were recorded at 0 h, 6 h, 12 h, and ultimate 24 h. CDX2 + IFN-γ vs. IFN-γ, ***p < 0.001, t test CDX2 is a transcription factor that has been shown with a more metastatic phenotype of gastric carcinoma to activate the Claudin-2 promoter in human intesti- cells [49], whereas CDX2 might act as a tumor suppres- nal epithelial cells and plays a crucial role in controlling sor during colorectal carcinogenesis [50]. These discrep - the balance between proliferation and differentiation of ancies led us to explore the fine regulation of CDX2 in intestinal epithelial cells [43–45]. An indirect pattern of both gastric and intestine epithelial cells as the C agA ZO-1 regulation by CDX2 via the MEK/ERK pathway H. pylori related primary lesion and secondary effect, was also verified in porcine intestinal epithelium [46]. respectively. CDX2 expression is significantly decreased in patients In summary, we demonstrated for the first time that with active UC, and it is involved in protection against chronic infection with CagA H. pylori exacerbates DSS-induced colitis [47, 48]. However, to date, the regu- DSS-induced UC-like colitis in mice. A series of in  vitro lation of mucosal inflammation by CDX2 as a triggering experiments confirmed the exosomal CagA-associated or compensative factor is rather unclear. Interestingly, a upregulation of Claudin-2 expression by the activation recent study indicated that CagA disrupts tight junctions of the transcription factor CDX2. The damaging role of by increasing the CDX2-dependent targeting of Clau- CagA in mucosal barrier integrity was verified by a series din-2 in gastric carcinoma cells and causes significant of functional experiments under IFN-γ-stimulating con- changes in the morphology and activity of these cells ditions. Although it is not clear whether intestinal epi- [49]. In our experiment, exosomal CagA upregulated thelial barrier dysfunction is a cause or a consequence of CDX2 expression and increased Claudin-2 expression to IBD, intestinal epithelial Claudin-2 can still be used as a impair the intestinal mucosal barrier. Similarities in these target to impair the integrity of the IBD intestinal barrier, results may be due to the infiltration of CagA exosomes which provides a new idea for IBD treatment. Further into either the gastric or colonic epithelium irrespective research is needed to determine whether such a mecha- of the different and complex intracellular environments. nism may also be involved in other extragastric diseases Moreover, increased CDX2 expression was associated caused by H. pylori infection. Guo  et al. Gut Pathogens (2022) 14:13 Page 9 of 13 Materials and methods without CagA H. pylori at a multiplicity of infection H. pylori culture of 100 for 2, 6, and 12  h. The conditioned media were + − CagA H. pylori and CagA H. pylori used in culture then collected from the coculture systems to isolate the and mouse studies were isolated from gastric specimens exosomes as described [37]. Briefly, cells and debris in of gastric ulcer patients during gastroscopy as described conditioned media were eliminated by successive cen- [37, 51]. In brief, bacteria were grown on Columbia blood trifugation (4 °C) at increasing speeds (300 g for 10 min, agar plates supplemented with antibiotics (10 mg/L van- 2000 g for 20 min, then 10,000 g for 30 min). The super - comycin, 5  mg/L cefsulodin, 5  mg/L amphotericin B, natant was next transferred to an ultracentrifuge tube 5  mg/L trimethoprim, and 10% sheep blood (Thermo and centrifuged at 100,000  g at 4  °C for 70  min twice Scientific R54008, Waltham, MA)) at 37 °C under micro - (Beckman Coulter, Indianapolis, IN). Exosome pellets aerophilic conditions (5% O , 10% CO , and 85% N ) for were resuspended in a small volume containing PBS for 2 2 2 3–4  days. After incubation for 3–4  days, the bacterial further analysis. The protein level in the exosomes was colonies were collected by scrape and then resuspended determined by the BCA protein assay (Cat.No.23235; in PBS. The bacterial concentration was calculated based Thermo Fisher, Waltham, MA). The morphologies, size on an optical density (OD) of 1 × 10 colony-forming distribution, and biomarkers (HSP70 and CD9) of the units (CFUs) at 660 nm. Identification of CagA H. pylori exosomes were examined using a transmission electron was confirmed using the complete sequence data of the microscopy (TECNAI G2 Spirit; FEI, Hillsboro, OR), H. pylori 16S rRNA gene from GenBank data (sequence dynamic light scattering with a particle and molecular ID AP017362) and positive biochemical test reactions for size analyzer (Zetasizer Nano ZS; Malvern Panalytical, oxidase, catalase, and urease. Malvern, Worcestershire, UK), and western blotting, respectively. Cell culture and cell‑bacteria coculture The human gastric epithelial cell line GES-1 and intes - Cell viability and migration assay tinal epithelial cell line NCM460 were continuously Cell proliferation was analyzed using a commercial cultured in Dulbecco’s modified Eagle’s medium (Sigma– CCK-8 assay kit (#C0038, Beyotime) and EdU detection Aldrich, USA) containing 10% fetal bovine serum (FBS) kits (RiboBio, Guangzhou, China). The wound scratch (Gibco, New York, USA), 100  IU/mL penicillin G and assay was performed to evaluate the migration ability. 100 μg/mL streptomycin (Sigma–Aldrich, USA) in a con- Polarized monolayers of NCM460 cells on transparent trolled humidified incubator at 37 °C with 5% CO . supports were washed twice with prewarmed PBS, and wounds were created by scraping the monolayers with a Reagents, plasmid construction, RNA interference 10  nm pipette tip. Media containing drugs (IFN-γ) was and transfection then applied. Twelve random wound points were chosen, The chemical reagents IFN-γ (cat. #PHC4033) was from and images were taken at time 0  h, 6  h, 12  h, and 24  h. Gibco (Houston, TX, USA). The recombinant His-CagA The wound closure area was measured using NIH ImageJ protein and the full-length CDX2 plasmid tagged with software. GFP were purchased from GeneChem Biotechnol- ogy Company (Shanghai, China). Short hairpin RNA (shRNA) for CDX2 and its corresponding negative TEER measurement control were also synthesized by GeneChem (Shang- To evaluate the barrier integrity of NCM460 cells, the hai, China). Transfections were conducted using Lipo- transepithelial electrical resistance (TEER) was meas- fectamine 2000 (Invitrogen) according to a previously ured. The initial TEER was tested before the cells were described protocol [52]. Knockdown efficiencies were seeded. Colon cells with and without treatment were determined by immunofluorescence and western blot - seeded in a Transwell membrane insert (12 mm diameter, ting, and RNAi sequence with relatively high results was 0.4  μm pore size, Corning) at a density of 7 × 10   cells/ selected for further experiment. The primer informa - well. Then, 200 μL and 500 μL medium was added to the tion of CDX2, ZO-1, and Claudin-2, as well as target- apical and basal compartments, respectively. Cecropin A ing sequences of CDX2, are listed in Additional file  1: (12.5 μg/mL) was added to the apical and basal compart- Table S1. ments. The TER values were measured every day by using an ohm-meter fitted with chopstick electrodes (Millipore ESR-2; Burlington, MA, USA). Before each test, the plates Exosome isolation and identification were placed at room temperature for 30  min. The TER To prepare exosomes from conditioned media, GES-1 was calculated by using the following equation: TEER (Ω  human gastric epithelium cells were cultured with or cm ) = (TEER − TER ) × 0.3. initial Guo et al. Gut Pathogens (2022) 14:13 Page 10 of 13 Luciferase assay 2) mice administrated H. pylori via gavage only (Hp, Cells were seeded into 96-well plates and grown to n = 10); 3) mice receiving PBS via gavage and DSS (DSS, approximately 80% confluency on the second day. Next, n = 10); and 4) mice receiving CagA H. pylori via gav- the relevant reporter plasmids and the PRL-TK reporter age and DSS (Hp + DSS, n = 10). were transiently cotransfected into the cells. After 48  h, The Hp-infected groups were administered 2.5 × 10 firefly luciferase activity and Renilla activity were deter - CFU CagA H. pylori by oral gavage once every other day mined. The ratio of firefly luciferase activity to Renilla seven times over 13 days to ensure a 100% infection rate. activity was regarded as indicating the activity of the gene At the same time, the mice in the corresponding control promoters. Each experiment was performed in triplicate. group received PBS by oral gavage as the control. All mice were fasted 12  h before and 4  h after each inocu- lation. CagA H. pylori colonization in the gastric epi- Immunofluorescence staining thelium was confirmed histopathologically using gastric Cells were grown on polyethyleneimine-coated cover- + tissues from the mice after 8  weeks of CagA H. pylori slips, washed with prewarmed phosphate-buffered saline infection with Giemsa and Warthin-Starry silver staining. (PBS), fixed in 4% paraformaldehyde for 15 min, permea - + For the colitis experiments after 8  weeks of CagA bilized with 0.5% Triton X-100 in PBS for 10 min, blocked H. pylori infection, the DSS and Hp + DSS groups were with 3% bovine serum albumin (BSA) solution for 1  h, subjected to three repeated cycles of 2% DSS (Sigma, St and incubated with anti-primary antibodies in 3% BSA at Louis, MO, USA) to induce chronic colitis (7 days/cycle) 4 °C overnight (antibody information listed in Additional as instructed [53], and each was separated by 7  days of file  2: Table  S2). Cells were then rinsed three times for regular water. During the process, the body weight, dis- 5  min with PBS and incubated with individual primary ease activity index (DAI) and general status of all mice antibodies in 3% BSA at 37  °C for 90  min. Alexa488 or were observed every three days. Specifically, DAI scoring Alexa555 anti-rabbit secondary antibodies (1:100, Inv- was measured as described previously (DAI = (Weight itrogen) were added for 1  h in the dark. Nuclei were loss score + Stool characters score + Hematochezia counterstained with DAPI (1:1000, sc-3598; Santa Cruz) score)/3) [54]. Mice were sacrificed on the seventh day in PBS at room temperature for 2  min, rinsed with PBS after returning to drinking water freely. Colon length three times for 3 min, and mounted with SlowFade Gold and the weight of spleen were measured. Colon tissues Antifade Reagent (S36942, Life Technologies). Images for histological analyses were dissected and immediately were obtained from a confocal microscope (TCSSP8, fixed with 4% paraformaldehyde. Leica Microsystems) equipped with an acousto-optic The colons were opened longitudinally and briefly beam splitter, a 405-nm laser (for DAPI), an argon laser cleansed with cold PBS. Tissues were then divided into (488 nm for Alexa 488), and a diode-pumped solid-state two equal sections. The proximal parts were fixed in (DPSS) laser (561 nm). 10% formalin for histological examination while the dis- tal parts were stored at -80  °C for protein extraction. Paraffin-embedded sections of the indicated colons were Quantitative real‑time RT–PCR and RNA array subjected to H&E staining and then examined by light Quantitative real-time RT–PCR (qRT–PCR) analyses microscopy. The histological scores were analyzed as were conventionally carried out. The primers used for the described previously [55]. qPCR of the referenced genes are shown in Additional file 1: Table S1. Protein extraction, immunoprecipitation, and western blot analysis Chronic colitis models of  CagA H. pylori‑infected mice Protein extracts were obtained from fresh cells and colon All animal experiments were performed in accord- tissues with PBS and in RIPA buffer (Thermo Scientific, ance with the ARRIVE Guidelines. The study was MA, USA) supplemented with protease inhibitor and conducted according to the guidelines of the Declara- phosphatase inhibitor (Roche, Welwyn Garden, Switzer- tion of Helsinki, and approved by the Experimental land). Supernatants were eluted in SDS sample buffer and Animal Ethics Committee of the Third Xiangya Hos - analyzed by SDS–PAGE. For immunoblotting, primary pital of Central South University (The Research Code antibodies are indicated in Additional file  2: Table  S2. #LLSC(LA)2018-039, 16 January 2018). Specific-path - Blots were then developed with ECL western blotting ogen-free (SPF) male C57BL/6 mice (4  weeks) were reagents (Pierce Biotechnology, Rockford, IL). Signal cultured in SPF conditions. Forty mice were randomly intensity was quantified with ImageJ (National Institutes divided into four groups: 1) mice receiving a standard of Health, Bethesda, MD). diet and normal drinking water (control, NC, n = 10); Guo  et al. Gut Pathogens (2022) 14:13 Page 11 of 13 Chromatin immunoprecipitation (ChIP) assay Additional file 6: Figure S4. Cell proliferation and viability assay using For ChIP, 1.5 × 10 cells were subjected to a two-step dual EdU and CCK-8. These results indicated the confluence of the NCM460 cell monolayer at the beginning, and the proliferation and survival of colonic cross-linking procedure based on previously described cells were not affected by either CagA exosomes or IFN-γ stimulation. methods [56]. In brief, protein-DNA complexes were immunoprecipitated with anti-CDX2 antibodies followed Acknowledgements by q-PCR using a BioRad CFX-96 quantitative thermo- The authors are grateful to the Endoscopic Unit of Third Xiangya Hospital of cycler and SsoFast EvaGreen Low-ROX qPCR Super- Central South University for providing clinic samples to isolate H. pylori strains. Mix (BioRad). For qPCR quantification, the following Authors’ contributions primer pairs are listed in Additional file  1: Table S1. Data YG, CX, and XL designed the study, analyzed, and interpreted the data, were analyzed according to the ΔΔCT method (Applied and wrote the manuscript. RG, TH and XZ contributed to the data acquisi- Biosystems). tion, analysis, and interpretation. RG and XX (Xiaoran Xie) performed all the bioinformatics analyses. YG, TH, XZ, JC, HL and XX (Xiujuan Xia) carried out the experiments. CX and XX (Xiujuan Xia) provided technical expertise and sup- port. All authors read and approved the final manuscript. Bioinformatic and statistical analyses Funding Claudin-2 promoter (−  2000  bp) sequences (H. sapiens) This work was supported by the National Science Foundation of China were downloaded from UCSC (http:// genome. ucsc. edu/). (82002614), the Natural Science Foundation of Hunan Province (2021JJ40935), The specific binding of transcription factors in gene pro - the Wisdom Accumulation and Talent Cultivation Project of the Third Xiangya Hospital of Central South University (YX202103), the Fundamental Research moters was predicted by the Jasper database (http:// jaspa Funds of Central South University (1053320183950), and the National Innova- r2016. gener eg. net/). Statistical analyses were performed tive Training Program of China (201310533059). using SPSS 19.0 software (version 19.0; SPSS Inc., Chi- Availability of data and materials cago, IL). The results are presented as the mean ± S.D., Not applicable. and two-group comparisons were evaluated using Stu- dent’s t test. Declarations Ethics approval and consent to participate Abbreviations All the animals received human care. The study protocol conformed to the H. pylori: Helicobacter pylori; CagA: Cytotoxin-associated gene A; T4SS: Type IV ethical guidelines of the 1975 Declaration of Helsinki and was approved by secretion system; TJ: Tight junction; IBD: Inflammatory bowel disease; CDX2: the Experimental Animal Ethics Committee of the Third Xiangya Hospital of Caudal related homeodomain transcription 2; ZO: Zonula occludens; GBP1: Central South University ( The Research Code #LLSC(LA)2018-039, 16 January Guanylate-binding protein 1; TEER: Transepithelial electrical resistance; DSS: 2018). Dextran sulphate sodium. Consent for publication Not applicable. Supplementary Information The online version contains supplementary material available at https:// doi. Competing interests org/ 10. 1186/ s13099- 022- 00486-0. The authors declare that they have no competing interests. Author details Additional file 1: Table S1. Primers and shRNAs used in this study. Department of Gastroenterology, Third Xiangya Hospital of Central South Additional file 2: Table S2. Antibodies for western blot, immunoprecipi- University, 138 Tongzipo Road, Changsha 410013, China. Depar tment tation, and immunofluorescence in this study. of Ophthalmology, The Second Xiangya Hospital, Central South University, Changsha 410008, China. Hunan Key Laboratory of Nonresolving Inflamma- Additional file 3: Figure S1. Confirmation of H.pylori colonization in tion and Cancer, Changsha 410013, China. gastric mucosa by Giemsa and silver staining in C57BL/6 mice. Stainings in H. pylori alone group and H. pylori+DSS group indicated that DSS had no Received: 22 September 2021 Accepted: 10 March 2022 effects on H. pylori colonization. scale bar, 20µm. Additional file 4: Figure S2. 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Gut PathogensSpringer Journals

Published: Mar 24, 2022

Keywords: CagA; Exosome; Colitis; Tight junction; Claudin-2; CDX2

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