Bacterial chemotaxis modulates host cell apoptosis to establish a T-helper cell, type 17 (Th17)-dominant immune response in Helicobacter pylori infection Annah S. Roliga, J. Elliot Carterb, and Karen M. Ottemannc,1 Departments of aMolecular, Cellular, and Developmental Biology and cMicrobiology and Environmental Toxicology, University of California, Santa Cruz, CA 95064; and bDepartment of Pathology, University of South Alabama College of Medicine, Mobile, AL 36688 Edited by Ralph R. Isberg, Howard Hughes Medical Institute/Tufts University School of Medicine, Boston, MA, and approved October 27, 2011 (received for review March 22, 2011) The host inflammatory response to chronic bacterial infections often of their contact with the pathogen, because the APCs produce dictates the disease outcome. In the case of the gastric pathogen cytokines that dictate the character of the adaptive immune re- Helicobacter pylori,hostinflammatory responses result in outcomes sponse. Dendritic cells interacting with H. pylori fuel the pro- that range from moderate and asymptomatic to more severe with liferation of particular T cells, including T helper cells, type 1 (Th1 concomitant ulcer or cancers. It was found recently that H. pylori cells) (8), CD25+FoxP3+ T-regulatory cells (T-regs) (8, 9), and T − chemotaxis mutants (Che ), which lack directed motility but colo- helper cells, type 17 (Th17 cells) (10). nize to nearly wild-type levels, trigger less host inflammation. We The inflammatory response to H. pylori includes all these T-cell used these mutants to observe host immune responses that resulted types. However, the roles of the Th17 and T-reg cell populations in reduced disease states. Here we report that these mutants are during H. pylori infections have been debated recently. The Th17 defective for early gastric recruitment of CD4+ T cells compared with cell is involved in promoting chronic inflammation (11, 12); the T- − wild-type infection. Furthermore, Che mutant infections lack the T- reg cell, in contrast, regulates host immune responses. Th17 and T- helper cell, type 17 (Th17) component of the immune response, as reg cells are developmentally related and exist in a delicate balance measured by cytokine mRNA levels in gastric tissue via intracellular (13) that can dictate the outcome of a bacterial infection (14). cytokine staining and immunofluorescence. We additionally find MICROBIOLOGY − Evidence suggests that H. pylori pathogenesis results primarily that a Che mutant infection results in significantly less host cell from the immune response, and thus understanding how this im- apoptosis than does wild-type infection, in accordance with previous mune response is initiated and controlled is critical. Currently it is observations that T-helper cell, type 17 responses in Citrobacter unknown if a Th17 response (12) or a T-reg response (9) underlies rodentium infections are driven by concomitant bacterial and apopto- the ineffective immune response to H. pylori. Therefore, we sought tic cell signals. We propose that bacterial chemotaxis allows H. pylori to understand better how H. pylori promotes gastritis by comparing to access a particular host niche that allows the bacteria to express or the host immune cell and cytokine responses to wild-type H. pylori − deliver proapoptotic host cell factors. This report indicates that che- and to a Che mutant. Our studies provide evidence that bacte- motaxis plays a role in enhancing apoptosis, suggesting bacterial rially driven interactions with host tissues alter the nature of the chemotaxis systems might serve as therapeutic targets for infections immune and pathological response generated during infection. whose symptoms arise from host cell apoptosis and tissue damage. Results and Discussion T regulatory cells | adaptive immunity | pathogenesis H. pylori Chemotaxis Increases Inflammation 2 mo After Inoculation. − As stated above, Che H. pylori cause milder inflammation than do nfection with the gastric pathogen Helicobacter pylori leads to wild-type infections after 3–6 mo of colonization (4). To determine Ichronic inflammation, or gastritis, in all individuals. This bac- whether bacterial chemotaxis affected inflammation earlier, we terium colonizes 50% of the world’s population and triggers a examined inflammation at the earliest time inflammation was de- wide range of disease severities; many infected individuals remain tectable, 2 mo after inoculation. For these experiments, we orally − asymptomatic, but others develop peptic or gastric ulcers, gastric infected mice with either wild-type H. pylori or an isogenic Che adenocarcinoma, or mucosa-associated lymphoid tumors (1). mutant lacking a central chemotaxis protein, CheY. H. pylori cheY The pathogenesis of H. pylori-induced inflammation is not well mutants have been characterized extensively and found to retain understood. Inflammation is promoted by both host factors (2) flagella and motility but to lack chemotaxis completely (5, 15). − and H. pylori factors, such as the proteins cytotoxin associated gene Che mutants have early mouse colonization defects but achieve A (CagA) (1, 2) and vacuolating cytotoxin A (VacA) (1, 3) and normal bacterial levels by 1 mo after inoculation (5, 16). All cheY bacterial chemotaxis (4). Chemotaxis is the bacterial ability to mutant-associated phenotypes can be complemented, indicating move toward beneficial environmental signals and away from that loss of cheY is responsible for the chemotaxis and animal- harmful ones. H. pylori genetically altered to lack chemotaxis colonization deficits (5, 15). Using standard inflammation grading − (Che ) retain flagella and motility but cannot migrate toward or that captures the number and distribution of lymphocytes, we away from environmental signals. In mouse models, these mutants found that inflammation was significantly lower in mice infected − have a marginal colonization defect (4–6) but induce less overall for 2 mo with Che H. pylori than in mice infected with wild-type H. − chronic inflammation (4). Specifically, Che mutants localize far pylori but was greater than in the no-H. pylori control (Fig. 1). from the epithelial surface and do not colonize the gastric glands robustly (4, 6), suggesting that chemotaxis-driven contact with epithelial cells, resident dendritic cells, or monocytes promotes the Author contributions: A.S.R. and K.M.O. designed research; A.S.R. and J.E.C. performed inflammatory response to H. pylori. research; A.S.R. and K.M.O. analyzed data; and A.S.R. and K.M.O. wrote the paper. Inflammation begins when resident monocytes and epithelial The authors declare no conflict of interest. cells detect injury or a pathogen such as H. pylori (7). Epithelial cells This article is a PNAS Direct Submission. secrete chemokines to recruit antigen-presenting cells (APCs) such 1To whom correspondence should be addressed. E-mail: [email protected]. as dendritic cells that will prime T cells (7). The newly recruited This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. APCs definetheimmuneresponsetoH. pylori based on the nature 1073/pnas.1104598108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1104598108 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 − We infected mice with either wild-type or Che H. pylori and allowed infections to proceed for 2 wk to 3 mo. The total T-cell populations (CD3+CD45+) in infected stomachs rose steadily from week 2 to week 5 and displayed a subtle trend for the wild − type to recruit more T cells than the Che mutant (Fig. S1A). Additionally, the percentage of CD4+ T cells remained at back- − ground levels in Che infections but increased significantly above background in wild-type H. pylori infections (Fig. S1B). T-cytotoxic cells (CD8+CD3+) never increased over the mock-infection con- − trol and were not different between the wild-type and Che infections (Fig. S1B), in agreement with previous reports that this Fig. 1. H. pylori chemotaxis promotes inflammation. Inflammation grade of T-cell type does not play a major role in H. pylori immunity (17). stomach sections taken from C57BL/6 mice 2 mo after infection with wild- When we extended our flow cytometric analysis to 3 mo after − − type or Che H. pylori SS1. n = 6 for wild-type and Che H. pylori infections; inoculation, we observed that the percentage of CD4+ T cells in − n = 3 for mock infections. Error bars represent SEM. At 2 mo after in- the Che infection had risen to match those in the wild-type in- oculation, there was a not a statistically significant difference between the log + − fection, suggesting that CD4 T-cell recruitment is delayed but not cfu/g stomach tissue for wild-type (6.29 ± 1.1) and Che H. pylori (5.72 ± 0.24). − fl *P < 0.05, Student t test. abrogated in Che infections (Fig. 2 A and B). To verify our ow cytometry result, we further used immunohistochemistry at 2 mo − after inoculation and indeed confirmed that wild-type and Che fi Overall scores at this time point are low, con rming that 2 mo after H. pylori recruited similar numbers of CD4+ cells at this later time fl inoculation is the earliest time point at which in ammation is (Fig. 2 C and D). However, we did observe slight differences in the fi + pathologically identi able. Bacterial numbers at 2 mo after in- distribution of CD4 cells between the two infection types, with 6 oculation in both infection groups were ∼1 × 10 cfu/g and did not wild-type infections bearing more widespread inflammatory cells − differ significantly from each other, similar to previous findings (4, (Fig. 2C). Overall, these findings show that early Che infections 5), suggesting that inflammatory differences are not caused by a recruit fewer CD4+ T cells than do wild-type infections. This dif- disparity in bacterial burden. Additionally, there was no correlation ference dissipates sometime between 5 and 8 wk after inoculation, within individual mice between bacterial colonization levels and but significant differences in the inflammation grading remain. The inflammation grade, as seen previously (4).