Background
Helicobacter pylori, a class I carcinogen inhabit in the stomach of approximately 50% of the human population while only 10–15% population either develop chronic gastritis or gastric adenocarcinoma or gastric mucosa-associated lymphoid tissue lymphoma [
1‐
4]. The underlying mechanisms governing the clinical outcome of
H. pylori infection are poorly understood. However, accumulated evidences suggested that differences in host immune responses, environmental factors as well as the virulence properties of
H. pylori strains may play important roles in determining the disease outcome. The most prominent
H. pylori virulence-associated determinant is the
cag pathogenicity island (PAI). It is a 40-kb genome segment that encodes the immunodominant protein
cagA and type IV secretion system, which serve to transfer the bacterial
cagA protein and other soluble factors such as peptidoglycans, to the cytoplasm of the host cells, known to play a key role in disease manifestation [
5‐
7]. Strains harboring the
cag PAI have been associated with more severe inflammatory responses than that induced by
cag
− strains [
6,
8,
9].
H. pylori specific host T cell response is predominantly a CD4+ T cell response polarized towards a T helper1 (Th1) phenotype [
10,
11].
H. pylori induced inflammation are associated with the production of pro-inflammatory cytokines and appear to be triggered partly by genes located within the
cag PAI [
6,
8,
12]. The gastric mucosal levels of the proinflammatory cytokine IL-1β, IL-6, IL-8 and TNF-α were increased in
H. pylori infected subjects [
13]. Earlier studies revealed that
H. pylori infection is also associated with a marked increased in cytokine IL-17 secretion from Th17 cells [
14]. Involvement of IL-17 has also been reported in various other chronic inflammatory conditions such as rheumatoid arthritis and multiple sclerosis [
15,
16]. Recently, Shi et al. suggested that
H. pylori infection induced a mixed Th1/Th17 response [
17]. In addition,
cagA and type IV secretion system are required for the induction of IL-17 responses in
H. pylori infection [
18]. Secretion of IL-17 led to induction of other inflammatory molecules required for the establishment of chronic inflammation [
19].
MMPs are a family of zinc dependent endopeptidases that play a crucial role in various pathological conditions including gastric ulcer [
20‐
22]. The activities of MMPs are regulated by their inhibitors (TIMPs), while their expressions are modulated by cytokines, growth factors, tumor promoters and transcription factors [
20,
23,
24]. Gelatinases B (MMP9) and stromelysin-1 (MMP3) are the two major inflammatory contributors of gastric pathology, collectively cleave a large array of matrix proteins [
25]. Accumulated evidences suggested that
H. pylori induced gastric inflammation with the upregulation of MMP9 and MMP3 in vivo [
26]. MMPs are either directly or indirectly produced by gastric epithelial cells via cytokine mediated cell signaling pathways [
24].
The gastrointestinal tract of human are colonized by various microorganisms which can be either commensalistic or pathogenic to human [
27]. The interplay among those organisms can lead to either attenuation or promotion of infection-induced pathology. For instance, C57BL/6 mice coinfection with a natural murine nematode parasite
Heligmosomoides polygyrus, attenuated gastric pathology induced by
H. felis [
28]. Attenuation of gastric pathology was associated with reduced expression of proinflammatory Th1 cytokine as well as with increased Th2 cytokine levels. Interaction between different bacterial species also determines diseases severity. Recently it has been documented that
H. pylori infection attenuated
Salmonella enterica serovar Typhimurium-induced colitis in C57BL/6 mice; this protective effect was associated with downregulation of the cecal Th17 response to
S. typhimurium [
29]. Even severity of the
H. pylori induced gastric pathology is also modulated by coinfection with other
Helicobacter species. Coinfection of enterohepatic Helicobacter species (EHS),
Helicobacter muridarum along with
H. pylori attenuated the
H. pylori induced gastric pathology in C57BL/6 mice [
30]. Moreover, coinfection of another EHS,
Helicobacter hepaticus with
H. pylori lead to more severe gastritis as well as increased production of IL-17 cytokine [
30]. Interestingly, it has also been reported that the interactions between different strains of
H. pylori also modulate gastric inflammation status [
31].
Surprisingly, in some African countries having lower economic status show high rate of
H. pylori infection but low level of gastric carcinoma incidence, widely known as African Enigma [
32,
33]. The reasons could be associated with diet, infection with other endemic parasites and degree of pathogenicity of different
H. pylori strains. We hypothesized that coinfection with a non pathogenic strain may provide protection against further infection of a virulent strain. The effect of coinfection on gastric inflammation, with different strains of
H. pylori with or without
cag Pathogenicity Island, has not been systematically studied yet. To investigate that, we established a coinfection in C57BL/6 mice using both
cag
− and
cag
+ strains of
H. pylori and measured the gastric inflammatory pathways. We address whether prior
cag
− strain infection alleviate gastric damage induced by
cag
+
H. pylori strain coinfection and the underlying host immunomodulatory mechanisms thereon. Here we for the first time documents that prior infection with
cag
−
H. pylori strain dampens the disease severity for further
cag
+ coinfection.
Discussion
Helicobacter pylori colonization and associated pathology is determined by a combination of pathogen virulence factors and host immune response [
5,
6,
38].
H. pylori infection induced a robust proinflammatory Th1 and Th17 response that are associated with gastric inflammation, atrophy, epithelial hyperplasia and dysplasia [
10,
17,
18,
39]. Moreover, mixed or coinfection of different Helicobacter species/strains determined the outcome of disease severity. In this context, Secka et al. reported that mixed infection with
cag
+ and
cag
− strains of
H. pylori lowers disease burden among the Gambian population [
31]. Furthermore, coinfection with enterohepatic
Helicobacter species can reduce
H. pylori induced gastric pathology in C57BL/6 mice through modulation of gastric Th1 and Th17 responses [
30]. In present study we have investigated whether
cag
+ and
cag
−
H. pylori coinfection induces gastric mucosal inflammatory response differ from single strain infection. The study also focuses whether coinfection has any modulatory effect on gastric ulcer severity compared to single strain infection. We previously reported that both SS1 and AM1 strains were capable to cause gastric inflammation although the severity of damage was more pronounced in SS1 infection [
26]. Although, the functionality of SS1
cag gene within mouse gastric tissue is a controversial, but its association with severe gastric inflammation is well established. Our current result suggested that
cag
+ strain (SS1) induced gastric pathology were significantly attenuated in mice that were earlier coinfected with
cag
− strain (AM1) and associated with modulation of Th17 and Treg cell responses.
It is reported that
H. pylori infection is associated with elevated Th1 cytokines [
10,
40]. Hence, we examined whether reduced gastric inflammation in AM1/SS1 coinfected mice has any correlation with Th1 cytokines level. We found that despite the reduced gastric inflammation pathology in AM1/SS1 infected mice, the expression of inflammatory Th1 cytokines IFN-γ, TNF-α and IL-1β in AM1/SS1 and SS1/AM1 are comparable (Fig.
5). Interestingly, significantly lower level of IL-17 was detected in AM1/SS1 coinfected group than SS1/AM1 coinfected group. Previous studies established the role of proinflammatory Th17 pathway in the development of
H. pylori induced gastric inflammation in mouse model and human [
39,
41]. Yun shi et al. suggested that both Th1 and Th17 cells mediated mucosal inflammation is important in
H. pylori infection and Th17/IL-17 pathway modulates Th1 cell responses [
17]. Th17 cell responses are induced earlier than Th1 cell responses [
17], implying that Th17 and Th1 cells promote inflammation differentially. It is known that active type IV secretion system is required for IL-17 secretion [
18]. We found that
cag
+ strain infection induced IL-17A secretion in mouse gastric tissues as well as spleen, while
cag
− infection did not. It seems to us that severe gastric inflammation in SS1/SS1 infected mice were mediated by both Th1 and Th17 responses while AM1/AM1 infection only by Th1 responses. We found that the level of Th1 cytokines IFN-γ, TNF-α and IL-1β in AM1/SS1 infected mice are comparable to SS1/AM1infected mice. In contrast, higher level of IL-17A was detected in SS1/AM1 mice than AM1/SS1 infected mice. Thus our results clearly indicate that attenuated gastric pathology in AM1/SS1 infected group is not due to reduced Th1 responses instead of reduced Th17 responses to AM1/SS1 infection. We conclude that the Th1 cytokine induced by prior AM1 infection particularly IFN-γ could also contribute in part to the downregulation of Th17 response induced by subsequent
cag
+ (SS1) infection because IFN-γ plays an inhibitory role towards Th17 cell activation [
42,
43]. Thus, AM1 infection released high level of IFN- γ in the gastric lumen that prevented the activation of Th17 response resulting protection against further
cag
+ infection.
Expression and secretion of different MMPs in
H. pylori infection have been postulated to be critically involved in the development of gastric ulcer. However, recent evidences suggest that apart from its well studied inflammatory and pathogenic functions, MMPs play a more complex and diverse role in ECM homeostasis, regulation of inflammation, arresting disease progression [
22]. Role of cytokines and growth factors in regulation of MMPs expression have been reported earlier under various pathological conditions [
22,
44]. IL-17 stimulated gastric epithelial cells to produce MMP9 and 3 that might be important in mediating gastric inflammation. However, a significantly lower level of MMP9 and 3 expressions were detected in AM1/SS1 coinfected mice compared to SS1 alone or SS1/AM1 coinfected group (Fig.
3). In line with our observation, it has been reported that MMP9 expression in the stomach following
H. pylori infection was significantly reduced when IL-17 is deficient or blocked [
17]. Moreover, recombinant IL-17A treatment increased MMP9 expression in vitro [
17]. Our results show that the level of IL-17 is significantly increased only in the mouse gastric tissues infected with SS1 strains of
H. pylori, suggesting that
cag PAI is required for the induction of IL-17 cytokine, and also indicates that the cells producing MMPs have responded to the increased IL-17 secretions. Our results also suggested that the reduced gastritis in AM1/SS1 infected mice may be is due to reduced activation of Th17/IL-17 pathway and subsequent downregulation of MMP9 and 3 expressions in AM1/SS1 infected group.
It is well established that natural regulatory T (Foxp3
+ Treg) cells suppress the host inflammatory responses during infection and thereby maintain physiological homeostasis of host immunity [
45‐
47]. Elevated numbers of Treg cells were reported in
H. pylori positive patients and
H. pylori infected mice gastric tissues [
48‐
50]. Moreover inhibition of Treg cells function by treatment with monoclonal antibody resulted increased expression of gastric proinflammatory cytokines that lead to severe gastritis in
H. pylori infected mice [
48]. CD4
+CD25
+ Treg cells from
H. pylori positive patients are more potent in the suppression of memory T cell responses [
51]. Treg mediated immune suppression is predominantly utilizes IL-10 and TGF-β that currently gain much attention [
45,
46]. Previously it has been reported that
H. pylori induced gastritis was suppressed by adoptive transfer of Treg cells harvested from IL-10-competent C57BL/6 donor mice, demonstrating that IL-10-dependent Treg cells play a crucial role in suppressing
H. pylori-induced gastric disease [
52]. Our results also showed that the number of gastric foxp3
+ cells as well as gastric IL-10 and TGF-β level were significantly higher in
H. pylori infected mouse gastric tissues (Figs.
5,
6). While, AM1/SS1 infected mice with attenuated gastritis have fewer foxp3
+ cells and lower level of gastric IL-10 and TGF-β. Hence we found a positive correlation between severity of gastritis and no of Foxp3
+ cells as well as IL-10 and TGF-β expression. Previous reports suggested that IL-10 and TGF-β can suppress inflammatory Th17 as well as Th1 responses [
53,
54]. So it is reasonable to postulate that prior AM1 infection creating an anti-inflammatory bias to further
H. pylori infection at the outset of coinfection, with relatively lower demand for Treg cells at more chronic time points because the Th1 and Th17 response to subsequent
H. pylori infection was suppressed by prior AM1 primed Treg cells.
We hypothesized that dendritic cells exposed to H. pylori may promote the preferential differentiation of naïve T cells into Treg cells. Those exposed dendritic cells then assist the differentiation of Treg cells as well as it lost its capability to further induce Th1 and Th17 responses upon subsequent H. pylori infection. Thus prior AM1 infected group showed reduced gastritis as its deficiency to induce Th17 response and probably stimulation of an anti-inflammatory bias by accumulation of AM1 sensitized dendritic and Treg cells within the gastric mucosa. In both SS1 and AM1 infection, primed Treg cells generated in gastric mucosa and these Treg cells provide protection against further infection of H. pylori by either directly or through cross reactivity. In contrast, prior SS1 infection cause an increase in the level of Th1 and Th17 responses are sufficient to do gastric damage. Irrespective of inhibitory role of SS1 primed Treg cells subsequent SS1 infection enjoy the benefit of existing inflammatory bias for further infection. However, earlier infection with AM1 helps in elicitation of AM1-primed Treg cells as well as less inflammatory bias through reduced secretion of Th1 cytokines. Subsequent infection of SS1 is prevented due to enrichment of AM1 primed Treg cells in the gastric mucosa that might provide protection through creating an anti inflammatory bias as well as by providing an un-hostile environment due to reduced inflammatory bias.
Authors’ contributions
SS and NG carried out study conception and design. NG, KK, PG carried out the experiments, participated in the analysis and interpretation of data and drafted the manuscript. AKM contributed to data analysis and edited the manuscript. All authors read and approved the final manuscript.