Helicobacter pylori colonizes half of the world’s population as the dominant member of the gastric microbiota resulting in a lifelong chronic infection. Host responses toward the bacterium can result in asymptomatic, pathogenic or even favorable health outcomes, however, mechanisms underlying the dual role of H. pylori as a commensal versus pathogenic bacterium are not well characterized. Recent evidence suggests mononuclear phagocytes are largely involved in shaping dominant immunity during infection mediating the balance between host tolerance and succumbing to overt disease. We combined computational modeling, bioinformatics and experimental validation in order to investigate interactions between macrophages and intracellular H. pylori. Global transcriptomic analysis on bone marrow-derived macrophages (BMDM) in a gentamycin protection assay at six time points unveiled the presence of three sequential host response waves: an early transient regulatory gene module followed by sustained and late effector responses. Kinetic behaviors of pattern recognition receptors (PRRs) are linked to differential expression of spatiotemporal response waves and function to induce effector immunity through extracellular and intracellular detection of H. pylori. We report that bacterial interaction with the host intracellular environment caused significant suppression of regulatory NLRC3 and NLRX1 in a pattern inverse to early regulatory responses. To further delineate complex immune responses and pathway crosstalk between effector and regulatory PRRs we built a computational model calibrated using time-series RNAseq data. Our validated computational hypotheses are that: 1) NLRX1 expression regulates bacterial burden in macrophages; and 2) early host response cytokines down-regulate NLRX1 expression through a negative feedback circuit. This paper applies modeling approaches to characterize the regulatory role of NLRX1 in mechanisms of host tolerance employed by macrophages to respond to and/or to co-exist with intracellular H. pylori.

During chronic H. pylori infection hosts develop complex immune responses to cope with bacterial persistence that result in a variety of outcomes ranging from peaceful co-existence to detrimental disease. Innate immune cells play a decisive role in host fate by enabling high bacterial loads and minimizing the immunological impact of this burden in the gastric mucosa. Our results investigate a series of hypotheses driven by computational and mathematical modeling and suggest one mechanism required by macrophages to ensure survival and intracellular H. pylori without overzealous inflammatory signaling.

We performed a time series RNAseq experiement on macrophages infected with H. pylori to gain a global perspective of host-pathogen interactions. Our data suggest that mechanisms of mononuclear phagocyte response to H. pylori occur sequentially and may depend on the location of the bacterium with respect to the host cell. Additionally, we found that H. pylori induces a unique regulatory gene module with an early transient peak during infection including the anti-inflammatory cytokine Il10.

Next we investigated how innate pattern recognition receptors (PRRs) may facilitate the recognition of H. pylori to subsequently induce classic molecular cascades that efficiently modulate immunity. H. pylori infection altered the expression of nearly all PRRs. An interesting finding from our transcriptomic profiling demonstrates that BMDM treated with H. pylori have significantly repressed transcripts for Nlrc3, Nlrp12 and Nlrx1, a subgroup of regulatory PRRs that function to attenuate inflammation.

We hypothesized that NLRX1 represents a central molecular checkpoint involved in the fate of bacterial burden and macrophage responses during infection and constructed a calibrated ODE-based model to investigate the roles of effector versus regulatory PRRs during H. pylori infection. Our modeling and simulation studies generated four computational hypotheses surrounding the immunomodulatory mechanisms of macrophage-NLRX1 during H. pylori infection. First, H. pylori rapidly induces an early wave response that is largely responsible for NLRX1 suppression in infected BMDM. Second, sustained inflammatory mediators are required for the induction of NLRX1 expression during infection. Third, the loss of NLRX1 results in a sharp increase of NF-κB and early cytokines but little changes are observed in sustained or late cytokines. Lastly, NLRX1-deficiency results in lower levels of intracellular H. pylori when compared to wild type simulations

Taken together, these efforts in characterizing innate immune mechanisms that reduce the negative impact of H. pylori infection on host fitness without directly affecting microbial burden may result in host-based precision medicine strategies that do not rely on anti-microbial treatment. Broadly, our model of host response to H. pylori infection represents an invaluable tool to study the mucosal immune system as a massively interacting information processing system.

Clostridium difficile infections are associated with the use of broad-spectrum antibiotics and result in an exuberant inflammatory response, leading to nosocomial diarrhea, colitis and even death. To better understand the dynamics of mucosal immunity during C. difficile infection (CDI) from initiation through expansion to resolution, we built a computational model of the mucosal immune response to the bacterium. The model was calibrated using data from a mouse model of C. difficile infection. The model demonstrates a crucial role of T helper 17 (Th17) effector responses in the colonic lamina propria and luminal commensal bacteria populations in the clearance of C. difficile and colonic pathology, whereas regulatory T (Treg) cells responses are associated with the recovery phase. In addition, the production of anti-microbial peptides by inflamed epithelial cells and activated neutrophils in response to C. difficile infection inhibit the re-growth of beneficial commensal bacterial species. Computational simulations suggest that the removal of neutrophil and epithelial cell derived anti-microbial inhibitions, separately and together, on commensal bacterial regrowth promote recovery and minimize colonic inflammatory pathology. Simulation results predict a decrease in colonic inflammatory markers, such as neutrophilic influx and Th17 cells in the colonic lamina propria, and length of infection with accelerated commensal bacteria re-growth through altered anti-microbial inhibition. Computational modeling provides novel insights on the therapeutic value of repopulating the colonic microbiome and inducing regulatory mucosal immune responses during C. difficile infection. Thus, modeling mucosal immunity-gut microbiota interactions has the potential to guide the development of targeted fecal transplantation therapies in the context of precision medicine interventions.

To further understand these cellular dynamics and the overall host response to the bacterium, we have generated a tissue-level computational model of C. difficile infection using ordinary differential equations (ODEs) to describe the experimentally observed dynamics from individual cells to the gut mucosal immune system. In addition to the immune response, the model incorporates the interactions between host, pathogen and commensal bacteria and can be customized to model other bacteria and associated conditions.

The model topology is shown in Fig. 1. The network represents the immune response to CDI in the colonic mucosa. The computational model is comprised of four compartments (lumen, epithelium, lamina propria and mesenteric lymph nodes) and 23 species whose interactions are described by 30 reactions. The resulting ordinary differential equations (ODE) utilize 49 parameters to describe the dynamics of the system. Parameter values were determined using data generated through a murine model of CDI. A time course study was performed to evaluate changes in immune cell composition following CDI. On days 1, 3, 4, 5, 7, 8, and 10 post-infection, colons and mesenteric lymph nodes (MLN) were collected, processed and assayed to determine alterations in immune cell subsets by flow cytometry conducted in two sets. In addition, colonic contents were collected and plated to measure the C. difficile population size.

The response to infection was observed to be Th17 dominant with a large neutrophilic influx in the colonic mucosa. The Th17 response peaked on day five post-infection and corresponded with the largest increase in macrophage accumulation. In contrast a regulatory response, characterized by the accumulation of CD4+CD25+Foxp3+ Treg cells, was detected between days 8 and 10, and preceded by a slight suppression between days 4 and 7 post-infection.

Sensitivity analyses demonstrate that the induction of a regulatory response decreases damage to the epithelium following CDI. Specifically, parameters controlling the production of tolerogenic dendritic cells and the plasticity of Th17 cells to become Treg cells are highly associated with decreased epithelial damage. However, the induction of regulatory responses can also slow C. difficile clearance and increase the overall length of infection, as the removal of pro-inflammatory cell types is largely associated with a larger C. difficile population.

CDI is associated with the use of broad-spectrum antibiotics, and microbiome reconstitution through fecal transplantation or probiotic treatment has gained some traction with varying degrees of success. The presence of C. difficile indirectly inhibits the regrowth of these commensal species associated with protective effects. The combination of elevated antimicrobial peptides and the susceptibility of a major constituent of the beneficial microbiome to these proteins suggest a strong probability that the host response to C. difficile is also inhibitory to the regrowth of beneficial commensal strains. The simulated removal of epithelial cell-related inhibition of commensal bacterial regrowth in the model allows for an increased amount of beneficial commensal species early in the infection, resulting in slightly decreased C. difficile levels through direct competitive effects on the population itself and indirectly through down-regulation of neutrophil and Th17 cell-mediated effector responses. Additionally, the return of microbial diversity would greatly reduce the capacity for the persisting C. difficile population to expand back to pathogenic levels, decreasing the rate of reoccurrence.

In conclusion, we have examined the time course of the immune response to C. difficile infection through a combination of experimental and computational approaches using an iterative systems biology cycle. We described the importance of maintaining a balance between effector and regulatory arms of the mucosal immune response in the clearance of pathogen and severity of infection. Specifically, the production of anti-microbial peptides may exacerbate disease and pathology, and prolong the CDI due to non-specific inhibition of commensal bacterial regrowth. Computational simulations supported the role of this inhibition on the disease severity with a virtual removal of neutrophil and epithelial cell derived anti-microbial inhibitions, separately and together, on commensal bacterial regrowth. The simulated shifts in host response behavior provide novel insights underlying the mechanisms of interaction between the mucosal immune system and the gut microbiota during CDI.

Zinc Deficiency Alters Host Response and Pathogen Virulence in a Mouse Model of Enteroaggregative Escherichia coli-Induced Diarrhea

Enteroaggregative Escherichia coli (EAEC) is increasingly recognized as a major cause of diarrheal disease globally. In the current study, we investigated the impact of zinc deficiency on the host and pathogenesis of EAEC. Several outcomes of EAEC infection were investigated including weight loss, EAEC shedding and tissue burden, leukocyte recruitment, intestinal cytokine expression, and virulence expression of the pathogen in vivo. Mice fed a protein source defined zinc deficient diet (dZD) had an 80% reduction of serum zinc and a 50% reduction of zinc in luminal contents of the bowel compared to mice fed a protein source defined control diet (dC). When challenged with EAEC, dZD mice had significantly greater weight loss, stool shedding, mucus production, and, most notably, diarrhea compared to dC mice. Zinc deficient mice had reduced infiltration of leukocytes into the ileum in response to infection suggesting an impaired immune response. Interestingly, expression of several EAEC virulence factors were increased in luminal contents of dZD mice. These data show a dual effect of dietary zinc in benefitting the host while impairing virulence of the pathogen. The study demonstrates the critical importance of zinc and may help elucidate the benefits of zinc supplementation in cases of childhood diarrhea and malnutrition.

In a recent study, we have shown that culturing EAEC in the presence of low levels of Zn greatly reduce virulence factor expression and biofilm formation 1. Additionally, in our previous study, mice fed a diet with reduced Zn (~3.2ppm) had greater growth deficits than mice supplemented with Zn daily. We therefore chose to test EAEC infection in mice fed a virtually zinc-free protein source defined diet (dZD) or same diet with 30ppm added Zn (dC). Using this diet (dZD), we demonstrate significantly lower levels of zinc in both blood serum and intestinal tissue (P<0.0001 dC vs. dZD). Additionally, for the first time, we observe that dZD mice consistently developed diarrhea when infected with EAEC (compared to no diarrhea in the dC+042 group) and increased weight loss over the first 3 days of infection compared with all other treatment groups (P<0.01).

Zinc has been shown to inhibit the growth of other enteric pathogens such as salmonellae, Shigella, and Vibrio in vitro 2. Additionally, we have previously shown an inhibitory effect of zinc on EAEC in vitro 1. In the current study, we were able to demonstrate in vivo, increased expression of several aggR-regulated EAEC virulence factors in the absence of dietary zinc. Dietary supplementation of zinc not only empowers host immunity 3, but also has effects on pathogen virulence factors and their upstream regulators which may decrease disease severity 1. In the current study, we show the combined effects of dietary zinc on both increasing the host immunity as well as decreasing the pathogenicity of EAEC. In summary, zinc is a critical micronutrient in the prevention and treatment of EAEC infection.

1. Medeiros P, Bolick DT, Roche JK, et al. The micronutrient zinc inhibits EAEC strain 042 adherence, biofilm formation, virulence gene expression, and epithelial cytokine responses benefiting the infected host. Virulence 2013;4.

2. Surjawidjaja JE, Hidayat A, Lesmana M. Growth inhibition of enteric pathogens by zinc sulfate: an in vitro study. Med Princ Pract 2004;13:286-9.

3. Fraker PJ, King LE. Reprogramming of the immune system during zinc deficiency. Annual review of nutrition 2004;24:277-98.

The development of gastritis during Helicobacter pylori infection is dependent on an activated adaptive immune response orchestrated by T helper (Th) cells. However, the relative contributions of the Th1 and Th17 subsets to gastritis and control of infection are still under investigation. To investigate the role of interleukin-21 (IL-21) in the gastric mucosa during H. pylori infection, we combined mathematical modeling of CD4+ T cell differentiation with in vivo mechanistic studies. We infected IL-21-deficient and wild-type mice with H. pylori strain SS1 and assessed colonization, gastric inflammation, cellular infiltration, and cytokine profiles. Chronically H. pylori-infected IL-21-deficient mice had higher H. pylori colonization, significantly less gastritis, and reduced expression of proinflammatory cytokines and chemokines compared to these parameters in infected wild-type littermates. These in vivo data were used to calibrate an H. pylori infection-dependent, CD4+ T cell-specific computational model, which then described the mechanism by which IL-21 activates the production of gamma interferon (IFN-γ) and IL-17 during chronic H. pylori infection. The model predicted activated expression of T-bet and RORγt and the phosphorylation of STAT3 and STAT1 and suggested a potential role of IL-21 in the modulation of IL-10. Driven by our modeling-derived predictions, we found reduced levels of CD4+ splenocyte-specific tbx21 and rorc expression, reduced phosphorylation of STAT1 and STAT3, and an increase in CD4+ T cell-specific IL-10 expression in H. pylori-infected IL-21-deficient mice. Our results indicate that IL-21 regulates Th1 and Th17 effector responses during chronic H. pylori infection in a STAT1- and STAT3-dependent manner, therefore playing a major role controlling H. pylori infection and gastritis.

This study leverages our published CD4+ T cell model (32) and cellular H. pylori model (15) to establish a chronic-H. pylori-specific CD4+ T cell differentiation model that allowed us to investigate the role of IL-21 in the maintenance of the T cell-mediated gastric mucosal responses to chronic H. pylori infection.

Thus, we combined computational modeling and mechanistic experimental studies in mice to dissect the effects of H. pylori infection on the intracellular pathways by which IL-21 modulates CD4+ T cell responses during chronic infection. IL-21 deficiency leads to increased H. pylori colonization in the mouse model. Knockout mice at later time points had significantly higher levels of H. pylori colonization than their wild-type littermates. IL-21 deficiency protects H. pylori- infected mice from chronic gastritis.

Inflammation is reduced in H. pylori-infected IL-21 -/- mice compared to the levels in their H. pylori-infected wild-type littermates. Levels of acute and chronic inflammation were scored for stomach tissue (in the corpus and antrum) at 3 months postinfection. (Figure 1. A) Total inflammation was scored on a scale of 0 to 12 (bars represent means, and error bars represent upper and lower interquartile ranges). (Figure 1. B) Representative sections of the gastric mucosa from 3 months postinfection are presented (200). (Figure 1. C) Flow cytometric analysis was performed on dissociated stomach tissue at 3 months postinfection (n = 8 per genotype). Percentages of neutrophils (Gr1CD11c), macrophages (CD11b+ Gr1-), CD4+ CD3+ T cells, CD8+ CD3+ T cells, and B cells (B220) were calculated in the live-cell gate from the H. pylori-infected mice. Bars and error bars represent means SEM. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. Results are representative of 3 independent experiments.

Our in silico and in vivo data suggest that IL-21 is a key cytokine for maintenance of both the Th1 and Th17 response during H. pylori infection. IL-21 deficiency leads to abrogated Th17 and Th1 effector responses. Il-21 contributes to Th17 differentiation and modulates Th1 responses upon infection with H. pylori.

The CD4+ T cell computational model predicts modulation of differentiation and maintenance by IL-21 in silico. (Figure 2. A) Sensitivity analysis of IL-21 over Th1- and Th17-related molecules showing positive or negative correlation to IL-21. (Figure 2. B) Effect of IL-21 deficiency on modulation of RORt, IL-17, IL-21, and STAT3 in the computed Th17 system. (Figure 2. C) Effect of IL-21 deficiency on modulation of T-bet and IFN- in the computed Th1 system. (Figure 2. D and E) Upregulation of RORγt, IL-17, and STAT3-P in a Th17 state with increasing concentrations of IL-21 in the wildtype system (Figure 2. D) but not in the IL-21-/- system (Figure 2. E) during a time course of 200 hours.

Furthermore, we provide novel evidence that IL-21 is required for the development of gastritis and control of the H. pylori bacterial burden, as well as for the modulation of T cell-derived IL-10 and phosphorylation of both STAT1 and STAT3.

Predictive value of the CD4+ T cell differentiation model. (Figure 3. A) Cartoon representation of IL-21 interaction within the CD4+ T cell subset. (Figure 3. B) Side-by-side comparison assessing the differences between the model’s predicted percentages of change between the wild-type and the IL-21-/- systems and the experimental results for wild-type and IL-21-/- mice.

These data together represent key knowledge that could help in the development of novel IL-21-centered immunotherapeutics for controlling infectious and immune-mediated diseases.

Helicobacter pylori are gram-negative, miroaerophilic bacteria responsible for a variety of gastro-duodenal pathologies in the developed and developing world [1]. H. pylori is thought to be indigenous to the human population and is well adapted to colonize and persist in the human stomach. Infection is generally asymptomatic not causing clinical symptoms of those infected people. However, a small percentage of infected individuals will ultimately develop duodenal ulcers and gastric cancer due to the inability of the host immune system to clear the infection.

Dozens of bacterial factors are involved in H. pylori molecular pathogenesis (i.e. flagella, urease, catalase, neutrophil-activating protein Nap-A, vacA and cagA). These proteins have revealed many aspects of the relationships between the bacteria, the gastric mucosal surface, and the final outcome of the disease. Two of the most studied virulence factors are vacuolotoxin A (VacA) and the cytotoxin-associated gene A (cagA). Secreted VacA triggers pore formation in the cell membrane, endolysomal trafficking modification, cellular vacuolation, immune cell apoptosis and cell inhibition [2, 3]. CagA is an effector protein injected into the gastric epithelial cells by a type IV secretion system encoded by the cag pathogenicity island (cagPAI). Once inside the host cells, it localizes under the point of bacterial attachment and interacts with the protein zonulin (ZO-1) and the junctional adhesion molecule (JAM) [4]. CagA is then phosphorylated on EPIYA repeats in its phosphotyrosine (PY) region induce secretion of interleukin 8 (IL-8) [5].

As shown in figure 1, the inflammatory response towards H. pylori is initiated through the interaction between the pathogen lipopolysaccharides (LPS) and the Toll-like receptors (TLR) expressed on gastric epithelial cells [6]. Once in the gastric lamina propria, H. pylori is mainly found inside macrophages where their interaction leads to macrophage activation and cytokine release [7]. Macrophages interact with T helper (Th) cells during infection and release cell-polarizing cytokines such as IL-17 [8]. In addition, H. pylori infection also involves neutrophils and increased antigen presenting activity of dendritic cells (DC) [9].

MIEP is basically focused on the characterization of the mechanisms underlying immune responses to enteric pathogens by integrating mathematical and computational modeling approaches with experimental data (Figure 2). The integration of modeling and experimental approaches provides unprecedented opportunities for systems-level knowledge discovery. Given the complexity of the host-H. pylori interactions at the systems level and the broad range of possible outcomes, our team has developed a computational model of the gastric mucosal immune response towards H. pylori infection. Currently, the model is able to predict the distinct time-dependent behavior of the three main CD4+ T cells (Th1, Th17 and iTreg) showing an increased Th17 response at the early stage of infection that switches to a Th1 predominance in the chronic phase of the infection.

1. Abdulrasheed A, Lawal OO, Abioye-Kuteyi EA, Lamikanra A: Antimicrobial susceptibility of Helicobacter pylori isolates of dyspeptic Nigerian patients. Tropical gastroenterology : official journal of the Digestive Diseases Foundation 2005, 26(2):85-88.

2. Amieva MR, El-Omar EM: Host-bacterial interactions in Helicobacter pylori infection. Gastroenterology 2008, 134(1):306-323.

3. Tanaka S, Mizuno M, Maga T, Yoshinaga F, Tomoda J, Nasu J, Okada H, Yokota K, Oguma K, Shiratori Y et al: H. pylori decreases gastric mucin synthesis via inhibition of galactosyltransferase. Hepato-gastroenterology 2003, 50(53):1739-1742.

4.Amieva MR, Vogelmann R, Covacci A, Tompkins LS, Nelson WJ, Falkow S: Disruption of the epithelial apical-junctional complex by Helicobacter pylori CagA. Science 2003, 300(5624):1430-1434.

5. Eaton KA, Kersulyte D, Mefford M, Danon SJ, Krakowka S, Berg DE: Role of Helicobacter pylori cag region genes in colonization and gastritis in two animal models. Infection and immunity 2001, 69(5):2902-2908.

6. Cullen TW, Giles DK, Wolf LN, Ecobichon C, Boneca IG, Trent MS: Helicobacter pylori versus the host: remodeling of the bacterial outer membrane is required for survival in the gastric mucosa. PLoS pathogens 2011, 7(12):e1002454.

7. Ito T, Kobayashi D, Uchida K, Takemura T, Nagaoka S, Kobayashi I, Yokoyama T, Ishige I, Ishige Y, Ishida N et al: Helicobacter pylori invades the gastric mucosa and translocates to the gastric lymph nodes. Laboratory investigation; a journal of technical methods and pathology 2008, 88(6):664-681.

8. Dong C: TH17 cells in development: an updated view of their molecular identity and genetic programming. Nature reviews Immunology 2008, 8(5):337-348.

9. Zhuang Y, Shi Y, Liu XF, Zhang JY, Liu T, Fan X, Luo J, Wu C, Yu S, Chen L et al: Helicobacter pylori-infected macrophages induce Th17 cell differentiation. Immunobiology 2011, 216(1-2):200-207.

Helicobacter pylori infection is the leading cause for peptic ulcer disease and gastric adenocarcinoma. Mucosal T cell responses play an important role in mediating H. pylori-related gastric immunopathology. While induced regulatory T (iTreg) cells are required for chronic colonization without disease, T helper (Th)1 effector responses are associated with lower bacterial loads at the expense of gastric pathology. Pigs were inoculated with either H. pylori strain SS1 or J99. Phenotypic and functional changes in peripheral blood mononuclear cell (PBMC) populations were monitored weekly, and mucosal immune responses and bacterial loads were assessed up to two months post-infection.

Both H. pylori strains elicited a Th1 response characterized by increased percentages of CD4⁺Tbet⁺ cells and elevated IFN-γ mRNA in PBMC. A subset of CD8⁺ T cells expressing Tbet and CD16 increased following infection. Moreover, a significant increase in perforin and granzyme mRNA expression was observed in PBMC of infected pigs indicating a predominant cytotoxic immune response. Infiltration of B cells, myeloid cells, T cells expressing Treg- and Th17-associated transcription factors, and cytotoxic T cells was found in the gastric lamina propria of both infected groups. Interestingly, based on bacterial reisolation data, strain SS1 showed greater capacity to colonize and/or persist in the gastric mucosa compared to strain J99.

This novel pig model of infection closely mimics human gastric pathology and presents a suitable avenue for studying effector and regulatory responses towards H. pylori described in humans.

To assess whether H. pylori infection affects the expansion of specific T cell subsets, we evaluated the expression of the main transcription factors involved in the regulation of CD4⁺ T cell phenotype: FOXP3 (iTreg and nTreg), Tbet (Th1) and RORγt (Th17). An elevated proportion of CD4⁺ T cells expressed the Th1-associated transcription factor Tbet in response to H. pylori (Fig. 1A). We observed a transient single peak at day 7 post-infection followed by a sustained increase in CD4⁺Tbet⁺ cells from day 28 to day 49 post-challenge in SS1-infected pigs. The same pattern was found in J99-infected pigs except that the response declined by day 49 post-challenge. The percentage of RORgt⁺CD4⁺ T cells was significantly different on days 21 post-infection for SS1 and days 28 and 49 post-infection in J99-infected pigs (Fig. 1C). There was a more notable decline in the expression of the Treg-associated transcription factor FOXP3 in CD4⁺ T cells from pigs infected with the strain SS1 on day 28 post-infection (Fig. 1D) which mirrored the increase in Tbet and coincided with increased transcripts of IFN-g mRNA in PBMC for both SS1 and J99-infected pigs (Fig. 1B). These results provided the first indication of a predominant Th1 response induced in our experimental model by H. pylori.

CD4⁺ T cells expressing the T cell lineage specific transcription factors (A) Tbet (Th1), (C) RORγt (TH17) and (D) FOXP3 (iTreg, nTreg) were detected by flow cytometry. IFN-γ mRNA levels in PBMC were measured by qRT-PCR (B). Symbols indicate statistical differences between either the H. pylori J99 (#) or SS1 (*) infected group to the control group and between both infected groups (&), n=8-9, mean±SEM, p≤0.05.

Furthermore, we demonstrate that H. pylori infection results in a significant increase of Tbet expressing cytotoxic CD8b⁺ T cells (Fig. 2A-B). The shift in Tbet expression was first detected on day 35 post-infection when on average 62% of cells had detectable amounts of this transcription factor. The percentage of CD8b⁺Tbet⁺ cells declined thereafter although towards the end of the study, on day 49 post-infection, there were still significant differences between infected and non-infected pigs. A closer analysis revealed the expansion of circulating CD8β⁺ T cells (Fig. 2C) and an increase of CD16 expression on those circulatingCTL (Fig. 2D) upon infection. Furthermore, CD3^-CD8a⁺ NK cells were significantly increased in blood of SS1-infected pigs on day 35 post-infection (Fig. 2E).

Expression of Tbet was assessed in peripheral CTL (CD8β⁺) on day 42 post-infection (A) and over time (B). Representative flow cytometry dot blots for non infected and H. pylori J99 and SS1 infected pigs are presented. Numbers indicate percentage of positive cells within the single cell population (A). The number of (C) CTL (CD8β⁺) and (E) NK cells (CD3^-CD8α⁺), were enumerated in PBMC over time. Numbers of cells per ml of blood were calculated by applying the percentage of immune cells obtained by flow cytometry to the concentration of cells in whole blood. The percentage of CD16 expressing CTL was assessed throughout the study (D). Gene expression levels of perforin (F), granzyme A (G) and granzyme B (H) in PBMC were analyzed over time. Symbols indicate statistical differences between either the H. pylori J99 (#) or SS1 (*) infected group to the control group and between both infected groups (&), n=8-9, mean±SEM, p≤0.05.

In concordance with the observed expansion of circulating cytotoxic T cells, we detected a significant upregulation in the expression of genes involved in the cytotoxic activity of CTL and NK cells, perforin, granzyme A and B (Fig. 2F-H). Overall our data suggest the initial induction of an IFN-g-producing Th1 response orchestrated by the transcription factor Tbet and executed by cytotoxic T cells.

Re-isolation of H. pylori from infected pigs was performed at the end of the study. Overall, H. pylori SS1 was recovered from the stomach of all pigs in that group, while H. pylori J99 could only be re-isolated from 8 out of 12 pigs. When looking at bacterial burden in different regions of the stomach we found that the percentage of re-isolation was consistently higher in the SS1-infected group than in the J99 group with the exception of the fundus-A sub-region which showed similar frequencies for both strains (Fig. 3B). Microscopic changes were present in the stomach of both infected groups and were characterized by significant expansion and development of organized lymphoid aggregates and diffuse leukocytic infiltration. Both strains of H. pylori induced organized lymphoid tissue in the stomach mucosa, which was more predominant in the cardiac region (Fig. 3A).

Representative images were taken from hematoxylin and eosin stained specimens collected from the stomach region cardiac B (CB) and pyloric A (PA) of non infected and infected pigs at 10× magnification (A). H. pylori J99 and SS1 were re-isolated from 6 stomach locations at 2 months post-infection. Re-isolation data is expressed as percentage (B).

In summary, our findings that H. pylori elicits Th1 and CTL responses in a novel pig model correlate well with its role as a facultative intracellular pathogen. Clinical and in vitro studies with human cells provide increasing evidence that cytotoxic immune responses play a crucial role in H. pylori pathogenesis. Our data demonstrates for the first time an increase in circulating CTL and NK cells peaking on day 35 post-infection which coincides with increased IFN-γ gene expression.

The exact role of CD8⁺ T cells and whether they contribute to the depletion of H. pylori from the gastric mucosa deserves further investigation. Findings from re-isolation and histopathology suggest that CD8+ T cell responses elicited upon infection might be ineffective in the elimination of bacteria but rather contribute to tissue damage. Furthermore, the infiltration of regulatory cells found in the stomach at least partially counteracts proinflammatory responses and contribute to bacterial persistence.

Here, we present the first pig model of H. pylori infection that corroborates in an experimental setting that the predominant Th1 response induced by the bacterium leads to the expansion of cytotoxic cells, including CTL and NK cells. The hallmark of the immune response to H. pylori in humans is the infiltration of Treg cells, neutrophils and Th1 cells. We have been able to reproduce these findings in our pig model, showing infiltration of myeloid cells and FOXP3⁺ T cells suggesting the presence of Tregs in the gastric mucosa. Furthermore, our model shows a strong systemic Th1 response followed by cytotoxic T cell responses. Similar to H. pylori mediated chronic gastritis in humans, bacteria are able to persist in the pig stomach but at the expense of lesion development. While the role of CD8⁺ T cells in mouse models of H. pylori has only been studied using immunodeficient mice lacking CD4⁺ T cells, our pig model provides a more suitable in vivo system to study cytotoxic immune responses towards H. pylori observed in humans and an ideal setting for testing new therapeutic approaches.

Helicobacter pylori are gram-negative, miroaerophilic bacteria responsible for a variety of gastro-duodenal pathologies in the developed and developing world [1]. H. pylori is thought to be indigenous to the human population and is well adapted to colonize and persist in the human stomach. Infection is generally asymptomatic not causing clinical symptoms of those infected people. However, a small percentage of infected individuals will ultimately develop duodenal ulcers and gastric cancer due to the inability of the host immune system to clear the infection.

Dozens of bacterial factors are involved in H. pylori molecular pathogenesis (i.e. flagella, urease, catalase, neutrophil-activating protein Nap-A, vacA and cagA). These proteins have revealed many aspects of the relationships between the bacteria, the gastric mucosal surface, and the final outcome of the disease. Two of the most studied virulence factors are vacuolotoxin A (VacA) and the cytotoxin-associated gene A (cagA). Secreted VacA triggers pore formation in the cell membrane, endolysomal trafficking modification, cellular vacuolation, immune cell apoptosis and cell inhibition [2, 3]. CagA is an effector protein injected into the gastric epithelial cells by a type IV secretion system encoded by the cag pathogenicity island (cagPAI). Once inside the host cells, it localizes under the point of bacterial attachment and interacts with the protein zonulin (ZO-1) and the junctional adhesion molecule (JAM) [4]. CagA is then phosphorylated on EPIYA repeats in its phosphotyrosine (PY) region induce secretion of interleukin 8 (IL-8) [5].

As shown in figure 1, the inflammatory response towards H. pylori is initiated through the interaction between the pathogen lipopolysaccharides (LPS) and the Toll-like receptors (TLR) expressed on gastric epithelial cells [6]. Once in the gastric lamina propria, H. pylori is mainly found inside macrophages where their interaction leads to macrophage activation and cytokine release [7]. Macrophages interact with T helper (Th) cells during infection and release cell-polarizing cytokines such as IL-17 [8]. In addition, H. pylori infection also involves neutrophils and increased antigen presenting activity of dendritic cells (DC) [9].

MIEP is basically focused on the characterization of the mechanisms underlying immune responses to enteric pathogens by integrating mathematical and computational modeling approaches with experimental data (Figure 2). The integration of modeling and experimental approaches provides unprecedented opportunities for systems-level knowledge discovery. Given the complexity of the host-H. pylori interactions at the systems level and the broad range of possible outcomes, our team has developed a computational model of the gastric mucosal immune response towards H. pylori infection. Currently, the model is able to predict the distinct time-dependent behavior of the three main CD4+ T cells (Th1, Th17 and iTreg) showing an increased Th17 response at the early stage of infection that switches to a Th1 predominance in the chronic phase of the infection.

1. Abdulrasheed A, Lawal OO, Abioye-Kuteyi EA, Lamikanra A: Antimicrobial susceptibility of Helicobacter pylori isolates of dyspeptic Nigerian patients. Tropical gastroenterology : official journal of the Digestive Diseases Foundation 2005, 26(2):85-88.

2. Amieva MR, El-Omar EM: Host-bacterial interactions in Helicobacter pylori infection. Gastroenterology 2008, 134(1):306-323.

3. Tanaka S, Mizuno M, Maga T, Yoshinaga F, Tomoda J, Nasu J, Okada H, Yokota K, Oguma K, Shiratori Y et al: H. pylori decreases gastric mucin synthesis via inhibition of galactosyltransferase. Hepato-gastroenterology 2003, 50(53):1739-1742.

4.Amieva MR, Vogelmann R, Covacci A, Tompkins LS, Nelson WJ, Falkow S: Disruption of the epithelial apical-junctional complex by Helicobacter pylori CagA. Science 2003, 300(5624):1430-1434.

5. Eaton KA, Kersulyte D, Mefford M, Danon SJ, Krakowka S, Berg DE: Role of Helicobacter pylori cag region genes in colonization and gastritis in two animal models. Infection and immunity 2001, 69(5):2902-2908.

6. Cullen TW, Giles DK, Wolf LN, Ecobichon C, Boneca IG, Trent MS: Helicobacter pylori versus the host: remodeling of the bacterial outer membrane is required for survival in the gastric mucosa. PLoS pathogens 2011, 7(12):e1002454.

7. Ito T, Kobayashi D, Uchida K, Takemura T, Nagaoka S, Kobayashi I, Yokoyama T, Ishige I, Ishige Y, Ishida N et al: Helicobacter pylori invades the gastric mucosa and translocates to the gastric lymph nodes. Laboratory investigation; a journal of technical methods and pathology 2008, 88(6):664-681.

8. Dong C: TH17 cells in development: an updated view of their molecular identity and genetic programming. Nature reviews Immunology 2008, 8(5):337-348.

9. Zhuang Y, Shi Y, Liu XF, Zhang JY, Liu T, Fan X, Luo J, Wu C, Yu S, Chen L et al: Helicobacter pylori-infected macrophages induce Th17 cell differentiation. Immunobiology 2011, 216(1-2):200-207.

Differentiation of CD4+ T cells into effector or regulatory phenotypes is tightly controlled by the cytokine milieu, complex intracellular signaling networks and numerous transcriptional regulators. We combined experimental approaches and computational modeling to investigate the mechanisms controlling differentiation and plasticity of CD4+ T cells in the gut of mice. Our computational model encompasses the major intracellular pathways involved in CD4+ T cell differentiation into T helper 1 (Th1), Th2, Th17 and induced regulatory T cells (iTreg). Our modeling efforts predicted a critical role for peroxisome proliferator-activated receptor gamma (PPARγ) in modulating plasticity between Th17 and iTreg cells. PPARγ regulates differentiation, activation and cytokine production, thereby controlling the induction of effector and regulatory responses, and is a promising therapeutic target for dysregulated immune responses and inflammation. Our modeling efforts predict that following PPARγ activation, Th17 cells undergo phenotype switch and become iTreg cells. This prediction was validated by results of adoptive transfer studies showing an increase of colonic iTreg and a decrease of Th17 cells in the gut mucosa of mice with colitis following pharmacological activation of PPARγ. Deletion of PPARγ in CD4+ T cells impaired mucosal iTreg and enhanced colitogenic Th17 responses in mice with CD4+ T cell-induced colitis. Thus, for the first time we provide novel molecular evidence in vivo demonstrating that PPARγ in addition to regulating CD4+ T cell differentiation also plays a major role controlling Th17 and iTreg plasticity in the gut mucosa.

To facilitate a comprehensive representation of the dynamics associated with the major pathways activated by cytokines which control CD4+ T cell differentiation and plasticity, we constructed an ordinary differential equation (ODE)-based computational model including cytokines, membrane receptors and transcription factors (Figure 1).

The signaling network illustrates network topologies associated with differentiation towards T helper (Th)1 (red shadow), Th2 (green shadow), Th17 (blue shadow) and induced regulatory T cells (iTreg, yellow shadow). The network is provided in Systems Biology Markup Language-compliant format.

Thus, we combined computational modeling and mouse adoptive transfer studies to gain a better mechanistic understanding of the modulation of CD4+ T cell differentiation and plasticity at the intestinal mucosa of mice. Our sensitivity analyses highlighted the importance of PPARγ in the regulation of Th17 to iTreg plasticity. Indeed, in vivo evidence demonstrates that PPARγ is required for the plasticity of Th17 promoting a functional shift towards an iTreg phenotype.

More specifically, the loss of PPARγ in silico caused upregulation of RORγt and IL-17 in Th17 cells (Figure 2B) and down-regulation of FOXP3 in iTreg cells (Figure 2D) compared to wild-type CD4+ T cells (Figure 2A and 2C). These results demonstrate that PPARγ exerts a regulatory role in CD4+ T cell differentiation from a naïve state to Th17 or iTreg cells. The transfer of CD4+ T cells lacking PPARγ in vivo resulted in significantly greater accumulation of IL-17-producing Th17 cells and lower levels of FOXP3+ iTreg cells in spleen, MLN and colonic LP of recipient mice (Figure 2E and 2F).

(A-D) Computational simulation of the effect of PPARγ deficiency on differentiation from a naïve state into either Th17 or iTreg phenotypes. (E) Th17 cell accumulation in spleens of recipients of wild-type versus PPARγ null CD4+ T cells. (F) Treg cell accumulation in spleen, mesenteric lymph nodes (MLN) and lamina propria (LP) of SCID recipient mice.

Furthermore, computational results show that following induction of Th17 and subsequent PPARγ activation, IL-17, STAT-3 and RORγt were dramatically downregulated, whereas FOXP3 was upregulated, thereby demonstrating a phenotypic switch from a Th17 to an iTreg phenotype (Figure 3A). To ensure that parameter space scan and time-course were linked and the changes in PPARγ were being observed in a time-dependent manner, a combination of both was run, reiterating the phenotype switch with increasing concentrations of PPARγ over time observing an upregulation of FOXP3 and a downregulation of IL-17, RORgt and STAT3-P (Figure 3B). To address this hypothesis, naïve CD4+ T cells were transferred in vivo into RAG2^-/- recipients. When clinical signs of disease and colitis appeared, a subset of mice was sacrificed and spleen, MLN and colons were extracted to examine Th17 and Treg levels (baseline results). After verifying the presence of Th17 cells in colon, MLN and spleen, half of the mice were received a daily treatment of 70 mg/kg of pioglitazone given orally to activate PPARγ (Figure 3C). Histopathological examinations also showed that colons from recipient mice treated with pioglitazone had a significantly lower lymphocytic infiltration and crypt hyperplasia than those from non-treated recipients. Untreated mice maintained a predominant Th17 response characterized by increased levels of CD4+ T cells expressing RORγt and IL-17A. In contrast, pioglitazone-treated mice not only recovered from colitis and its associated weight loss, but also showed a switch from a predominant Th17 into an iTreg phenotype characterized by increased expression of FOXP3 and decreased IL17-A and RORγt in CD4+ T cells of the colonic LP and MLN (Figure 3D and 3E). This data supports the in silico prediction that activation of PPARγ in Th17 cells favors differentiation into iTreg cells, which facilitates colonic tissue reconstitution and recovery from disease.

(A) Computer simulation illustrating a down-modulation of IL-17, STAT3, RORγt and upregulation of FOXP3 in a differentiated Th17 cell following PPARγ activation. (B) Combination of time-course and PPARγ concentration scan to assess changes of IL-17, STAT3, RORγt and FOXP3 over time. (C) Experimental design for the validation of the model prediction. (D-E) Accumulation of iTreg and Th17 cells in the mesenteric lymph nodes (MLN) and colonic lamina propria (LP) of recipient mice.

In summary, we demonstrate for the first time that activation of PPARγ results in reprogramming of the CD4+ T cell molecular pathways that control the Th17 phenotype, leading to the induction of an iTreg phenotype. This phenotype switch is associated with protection from CD4+ T cell-induced colitis during adoptive transfer experiments in mice. Thus, the balance between Th17 and Treg cells helps delineate the outcome of immunological processes from effector inflammation to regulatory tolerance. Our modeling approaches allowed us to narrow the design of experiments and to better understand the molecular mechanisms of action controlling CD4+ differentiation. This new mechanistic knowledge is broadly applicable to the development of immune therapeutics for infectious, allergic and immune-mediated diseases. More specifically, we propose that PPARγ is a promising therapeutic target for chronic inflammatory and infectious diseases where Th17 cells contribute to the gut immunopathogenesis.

The population of CD4+ T cells is functionally and phenotypically heterogeneous consisting of at least four subsets involved in coordinating various aspects of adaptive immunity. Upon antigenic stimulation by antigen-presenting cells, naïve CD4+ T cells (Th0) expand and differentiate into at least three effector cell subsets referred to as Th1, Th2, Th17, and induced regulatory T (iTreg) cells (Figure 1). In addition to the four Th phenotypes studied and modeled in this project other CD4+ T cell phenotypes have been characterized, including transforming growing factor β (TGF-β)-producing CD4+ T cells (Th3), IL-10-producing CD4+ T cells (Tr1), IL-9-producing CD4+ T cells (Th9) and T follicular helper (Tfh) cells located in the follicular regions of lymph nodes and spleen. Signaling pathways controlling fates on these phenotypes are closely connected to the four core subsets (Th1, Th2, Th17 and iTreg) (Figure 2). Each of these phenotypes is characterized by distinct effector and regulatory functions, which are regulated by a signature pattern of cytokines and multiple transcription factors. The signaling pathways that lead to these four predominant fates are cross-regulated via feedback loops that facilitate a balanced immune response to pathogens or abnormal cells while avoiding chronic inflammation and autoimmunity.

The signaling network illustrates network topologies associated with differentiation towards T helper (Th)1 (red shadow), Th2 (green shadow), Th17 (blue shadow) and induced regulatory T cells (iTreg, yellow shadow). The network is provided in Systems Biology Markup Language-compliant format. We present for the first time a mathematical and computational model built upon the current paradigms of molecular interactions that occur in CD4+ T cells. This model will help us to elucidate the regulatory mechanisms underlying CD4+ T cell differentiation, identify novel putative CD4+ T cell subsets, and study the dynamics of Th cell differentiation. This is a first step toward a more comprehensive understanding of the dynamics of the CD4+ T cell differentiation process at the systems level. Thus, we are describing activation pathways by phenotype, as well as the inhibitory mechanisms that lead to the induction or suppression of a CD4+ T cell phenotype. Generating a mathematical model usually is comprised of three steps: first, a translation from the literature into a structural network is needed. The architecture of the model has to be assembled based on literature fates. Secondly, data extracted from the literature and data generated by our laboratory is inserted in the model to adjust the dynamics of the model and ensure the correct trends and behaviors of different molecules in the model. This process is known as ‘parameter estimation’. Once the parameters are set, a quality control check is needed to guarantee that signaling pathways are being activated promptly at the correct time with the right signal. At this point, the model is ready to start running in silico experimentation and generating predictions with the right initializations. Ultimately, computational results will be generated, in vitro and in vivo validation studies are performed and the data generated in those studies is used to re-calibrate the model, using ‘parameter estimation’ again, thus closing and completing the modeling process. Once all the relationships between molecules were set, they were incorporated in the CellDesigner diagram representing a single CD4+ T cell. This diagram represents the cellular response of one CD4+ T cell activating and inhibiting reaction that take place in three different places: the extracellular environment, the cytoplasm and the nucleus space. To model CD4+ T cell differentiation, hill function and mass action equations were used. While Hill Coefficient allowed us to quantify the effect of a ligand binding a macromolecule through cooperative binding, mass action laws can represent dynamic equilibriums for elementary reactions, considering products as a proportion of the participating molecules in the reaction. An example can be found below:

The parameter estimation computational approach was used to determine the unknown constants driving the dynamics of the model. Briefly, we used the Particle Swarm Optimization (PSO) algorithm to obtain computational values for our model parameters in order to fit our experimental data to the model. PSO is a global search algorithm and thus depends only minimally on the initial guess of each parameter and therefore avoids the subjective estimation caused by initial guesses in local methods as Levenberg-Marquardt. The results on the parameter estimation using Particle Swarm shows a good fitting between the experimental data and the values computationally estimated by COPASI with reduced weighted error (Table 1 and Figure 3). Moreover, we could estimate the parameters for the complete model with the parameter estimation task (Table 2).

A species is fitted computationally using experimental data and simulation algorithms. The objective value is the value that COPASI targets based on the experimental data and the computational simulation.
IL-17 and FOXP3 were fitted by COPASI using the ParticleSwarm algorithm. The fitted value (dark blue and pink dots) could reproduce the behavior of the measured value (red and light blue dots). The weighted error (green dots) is around 0, indicating that the fitting has been performed successfully. Once this step is completed, quality control is performed. Using the proper initialization given by literature, the system is induced to the four phenotypes and checked to reproduce the correct up- and downregulation of specific molecules. These four phenotype checks are the result of our CD4+ T cell modeling efforts after calibration and they provide evidence that our computational and mathematical model is capable of reproducing the behaviors of the four CD4+ T cell phenotypes in terms of cytokines, inductors and transcription. In addition, we demonstrate that the calibration process has been run successfully and the dynamics of the CD4+ T cell differentiation network model are adjusted to mimic immunological behaviors characteristic of each phenotype (Figure 4).

1. A fitted specie is fitted computationally using experimental data and simulation algorithms
2. The objective value is the value that COPASI targets based on the experimental data and the computational simulation

The addition of increasing amounts of IL-12, IL-18 and IFN-γ (Th1), IL-4 (Th2), IL-6 and TGF-β (Th17) or TGF-β alone (iTreg) as external stimuli in the system resulted in increasing amounts of related molecules for each phenotype. After passing quality control, the CD4+ T cell computational model was able to predict different trends summarized in Table 3.

The CD4+ T cell differentiation model predicted behaviors 1-4 at a post-calibration stage. These predictions are the results of in silico experimentation using scans, time-courses and loss-of-function approaches. The CD4+ T cell differentiation model is available in BioModels.net here and on our website. This model has been curated by Biomodels.net and the ODEs are also available in SBML format through biomodels.net This modeling work was initiated in January 2009, using a logical model for CD4+ T cell differentiation. Using the ODE-based approach, the model has evolved dramatically. The following is a list of archived CD4+ T cell model releases. Please click on individual releases for more details. The ordinary differential equations set can be found here. The following is a list of archived CD4+ T cell model releases. Please click on individual releases for more details.
The primary MIEP Team member responsible for maintaining this model is Adria Carbo at the Virginia Bioinformatics Institute. Please contact Adria with any questions or comments. For the latest release, The model is available for download in CellDesigner xml format. We have tested that the model is compatible with Cell Designer 4.3.

• Eighth, the latest, release on Feburary 5th, 2014
• Seventh, release on October 10th, 2012
• Sixth release on August 3rd, 2012
• Fifth release on April 21st, 2012
• Fourth release on Jan 14th, 2012
• Third release on September 7th, 2011
• Second release on June 7th, 2011
• First release on March 27th, 2011

T helper (Th) cells play a major role in the immune response and pathology at the gastric mucosa during Helicobacter pylori infection. There is a limited mechanistic understanding regarding the contributions of CD4+ T cell subsets to gastritis development during H. pylori colonization. We used two computational approaches: ordinary differential equation (ODE)-based and agent-based modeling (ABM) to study the mechanisms underlying cellular immune responses to H. pylori and how CD4+ T cell subsets influenced initiation, progression and outcome of disease. To calibrate the model, in vivo experimentation was performed by infecting C57BL/6 mice intragastrically with H. pylori and assaying immune cell subsets in the stomach and gastric lymph nodes (GLN) on days 0, 7, 14, 30 and 60 post-infection. Our computational model reproduced the dynamics of effector and regulatory pathways in the gastric lamina propria (LP) in silico. Simulation results show the induction of a Th17 response and a dominant Th1 response, together with a regulatory response characterized by high levels of mucosal Treg) cells. We also investigated the potential role of peroxisome proliferator-activated receptor γ (PPARγ) activation on the modulation of host responses to H. pylori by using loss-of-function approaches. Specifically, in silico results showed a predominance of Th1 and Th17 cells in the stomach of the cell-specific PPARγ knockout system when compared to the wild-type simulation. Spatio-temporal, object-oriented ABM approaches suggested similar dynamics in induction of host responses showing analogous T cell distributions to ODE modeling and facilitated tracking lesion formation. In addition, sensitivity analysis predicted a crucial contribution of Th1 and Th17 effector responses as mediators of histopathological changes in the gastric mucosa during chronic stages of infection, which were experimentally validated in mice. These integrated immunoinformatics approaches characterized the induction of mucosal effector and regulatory pathways controlled by PPARγ during H. pylori infection affecting disease outcomes.

Given the complexity, nonlinearity and abundance of feedback loops in mucosal immune responses to H. pylori and to facilitate a better understanding of the mechanisms underlying such immune responses at the systems level, we constructed a SBML network model depicting the major effector and regulatory pathways evoked during H. pylori infection (Figure 1).

In line with our ODE-results, we observed in the ABM that in the GLN, Th1 cells peaked on day 30 post infection and remained at high levels with fairly constant values throughout the rest of the infection period (Figure 2A, 2G). Th17 responses were induced in the GLN and later detected in the LP, together with a Treg cell response that persisted over time in both gastric LP (Figure 2B, 2C) and GLN (Figure 2H, 2I). Regarding Th17 cells, these simulations depicted the immunoregulatory role of PPARg in the myeloid subset since we observed significant differences in enhanced Th17 responses in the myeloid cell-specific PPARg knockout model when compared to the wild-type. Th17 cell numbers were also significantly higher in the T cell-specific PPARg knockout model when compared to the wild-type model in both LP (Figure 2B and 2E) and the GLN (Figure 2H and 2K). T cell-specific PPARg deficiency significantly impaired the expansion of the iTreg cell compartment starting at day 30 and showed an oscillatory behavior and significant differences until day 60 in the gastric LP (Figure 2C and 2F).

We extended our modeling approaches to determine which are the main factors involved in gastric lesion development during H. pylori infection by using sensitivity analysis. Our results using ABM showed how at the early stage of infection (up to week 2 post-challenge), the epithelial cell damage is mainly caused by the bacterium (Figure 3A). Interestingly, we observed a trend towards Th1 and Th17 cells triggering epithelial cell damage starting 3 weeks post-infection. At the chronic phase of the infection our results showed a dominant role of Th1 and Th17 effector cells in inducing epithelial cell damage (Figure 3A). H. pylori induced epithelial cell damage throughout the infection. However, at a later infection stage, the induction of damaged epithelial cells by the effector Th1 and Th17 phenotypes overshadowed the effect of H. pylori itself. Of note, sensitivity analysis performed in the deterministic model at day 60 post-infection also showed similar results (Figure 3B). Going one step further from the model prediction, we hypothesized that the effector T cell response and not the bacterium itself is the main cause of epithelial cell damage during the chronic phase of H. pylori infection. Validating this hypothesis and using wild-type mice that were infected with H. pylori, immunophenotyping results showed a pronounced increase of IL-17A- (Figure 3C) and IFNg-producing cells (Figure 3D) in the gastric LP after 30 and 60 days post-infection. Metronidazole treatment, being an approach to eliminate the bacteria from the stomach at day 30, did not affect effector cytokine expression. These results suggested that effector T cell responses are implicated in lesion development during infection as showed in a cartoon model representation, highlighting the involvement of DC, T cells and macrophages on the formation of gastric lesions in the LP is shown in Figure 3E.

In summary, we combined computational modeling approaches and mouse challenge studies to investigate how CD4+ T cells and other immune cell subsets are distributed in the gut mucosa during H. pylori infection. Our model simulated T cell responses to H. pylori by using both platforms: ODE and ABM. Our modeling efforts predicted higher levels of effector responses in both the LP and the GLN when deleting PPARg, thus highlighting the role of PPARg activation as a potential mechanism for modulating CD4+ T cell responses during bacterial infection and positioning PPARg as a candidate for immunotherapeutics development. Future studies will more fully realize the potential of multiscale modeling to understand mucosal immunity.

a) Mei MH, R.; Zhang, X.; Bisset, K.; Eubank, S.; Hoops, S.; Marathe, M.; Bassaganya-Riera, J.; : ENISI Visual, an Agent-based Simulator for Modeling Gut Immunity. IEEE International Conference on Bioinformatics and Biomedicine (BIBM) 2012.

b) Mei Y, Carbo, A., Hontecillas, R., Bassaganya-Riera,J. : ENISI SDE: A novel web-based stochastic modeling tool for computational biology. 2013 IEEE International Conference on Bioinformatics and Biomedicine 2013.

c) Wendelsdorf KV, Alam M, Bassaganya-Riera J, Bisset K, Eubank S, Hontecillas R, Hoops S, Marathe M: ENteric Immunity SImulator: a tool for in silico study of gastroenteric infections. IEEE transactions on nanobioscience 2012, 11(3):273-288.

Helicobacter pylori are gram-negative, miroaerophilic bacteria responsible for a variety of gastro-duodenal pathologies in the developed and developing world [1]. H. pylori is thought to be indigenous to the human population and is well adapted to colonize and persist in the human stomach. Infection is generally asymptomatic not causing clinical symptoms of those infected people. However, a small percentage of infected individuals will ultimately develop duodenal ulcers and gastric cancer due to the inability of the host immune system to clear the infection.

Dozens of bacterial factors are involved in H. pylori molecular pathogenesis (i.e. flagella, urease, catalase, neutrophil-activating protein Nap-A, vacA and cagA). These proteins have revealed many aspects of the relationships between the bacteria, the gastric mucosal surface, and the final outcome of the disease. Two of the most studied virulence factors are vacuolotoxin A (VacA) and the cytotoxin-associated gene A (cagA). Secreted VacA triggers pore formation in the cell membrane, endolysomal trafficking modification, cellular vacuolation, immune cell apoptosis and cell inhibition [2, 3]. CagA is an effector protein injected into the gastric epithelial cells by a type IV secretion system encoded by the cag pathogenicity island (cagPAI). Once inside the host cells, it localizes under the point of bacterial attachment and interacts with the protein zonulin (ZO-1) and the junctional adhesion molecule (JAM) [4]. CagA is then phosphorylated on EPIYA repeats in its phosphotyrosine (PY) region induce secretion of interleukin 8 (IL-8) [5].

As shown in figure 1, the inflammatory response towards H. pylori is initiated through the interaction between the pathogen lipopolysaccharides (LPS) and the Toll-like receptors (TLR) expressed on gastric epithelial cells [6]. Once in the gastric lamina propria, H. pylori is mainly found inside macrophages where their interaction leads to macrophage activation and cytokine release [7]. Macrophages interact with T helper (Th) cells during infection and release cell-polarizing cytokines such as IL-17 [8]. In addition, H. pylori infection also involves neutrophils and increased antigen presenting activity of dendritic cells (DC) [9].

MIEP is basically focused on the characterization of the mechanisms underlying immune responses to enteric pathogens by integrating mathematical and computational modeling approaches with experimental data (Figure 2). The integration of modeling and experimental approaches provides unprecedented opportunities for systems-level knowledge discovery. Given the complexity of the host-H. pylori interactions at the systems level and the broad range of possible outcomes, our team has developed a computational model of the gastric mucosal immune response towards H. pylori infection. Currently, the model is able to predict the distinct time-dependent behavior of the three main CD4+ T cells (Th1, Th17 and iTreg) showing an increased Th17 response at the early stage of infection that switches to a Th1 predominance in the chronic phase of the infection.

1. Abdulrasheed A, Lawal OO, Abioye-Kuteyi EA, Lamikanra A: Antimicrobial susceptibility of Helicobacter pylori isolates of dyspeptic Nigerian patients. Tropical gastroenterology : official journal of the Digestive Diseases Foundation 2005, 26(2):85-88.

2. Amieva MR, El-Omar EM: Host-bacterial interactions in Helicobacter pylori infection. Gastroenterology 2008, 134(1):306-323.

3. Tanaka S, Mizuno M, Maga T, Yoshinaga F, Tomoda J, Nasu J, Okada H, Yokota K, Oguma K, Shiratori Y et al: H. pylori decreases gastric mucin synthesis via inhibition of galactosyltransferase. Hepato-gastroenterology 2003, 50(53):1739-1742.

4.Amieva MR, Vogelmann R, Covacci A, Tompkins LS, Nelson WJ, Falkow S: Disruption of the epithelial apical-junctional complex by Helicobacter pylori CagA. Science 2003, 300(5624):1430-1434.

5. Eaton KA, Kersulyte D, Mefford M, Danon SJ, Krakowka S, Berg DE: Role of Helicobacter pylori cag region genes in colonization and gastritis in two animal models. Infection and immunity 2001, 69(5):2902-2908.

6. Cullen TW, Giles DK, Wolf LN, Ecobichon C, Boneca IG, Trent MS: Helicobacter pylori versus the host: remodeling of the bacterial outer membrane is required for survival in the gastric mucosa. PLoS pathogens 2011, 7(12):e1002454.

7. Ito T, Kobayashi D, Uchida K, Takemura T, Nagaoka S, Kobayashi I, Yokoyama T, Ishige I, Ishige Y, Ishida N et al: Helicobacter pylori invades the gastric mucosa and translocates to the gastric lymph nodes. Laboratory investigation; a journal of technical methods and pathology 2008, 88(6):664-681.

8. Dong C: TH17 cells in development: an updated view of their molecular identity and genetic programming. Nature reviews Immunology 2008, 8(5):337-348.

9. Zhuang Y, Shi Y, Liu XF, Zhang JY, Liu T, Fan X, Luo J, Wu C, Yu S, Chen L et al: Helicobacter pylori-infected macrophages induce Th17 cell differentiation. Immunobiology 2011, 216(1-2):200-207.

Clostridium difficile is an anaerobic bacterium that has re-emerged as a facultative pathogen and can cause nosocomial diarrhea, colitis or even death. Peroxisome proliferator-activated receptor (PPAR) γ has been implicated in the prevention of inflammation in autoimmune and infectious diseases; however, its role in the immunoregulatory mechanisms modulating host responses to C. difficile and its toxins remains largely unknown. To characterize the role of PPARγ in C. difficile-associated disease (CDAD), immunity and gut pathology, we used a mouse model of C. difficile infection in wild-type and T cell-specific PPARγ null mice. The loss of PPARγ in T cells increased disease activity and colonic inflammatory lesions following C. difficile infection. Colonic expression of IL-17 was upregulated and IL-10 downregulated in colons of T cell-specific PPARγ null mice. Also, both the loss of PPARγ in T cells and C. difficile infection favored Th17 responses in spleen and colonic lamina propria of mice with CDAD. MicroRNA (miRNA)-sequencing analysis and RT-PCR validation indicated that miR-146b was significantly overexpressed and nuclear receptor co-activator 4 (NCOA4) suppressed in colons of C. difficile-infected mice. We next developed a computational model that predicts the upregulation of miR-146b, downregulation of the PPARγ co-activator NCOA4, and PPARγ, leading to upregulation of IL-17. Oral treatment of C. difficile-infected mice with the PPARγ agonist pioglitazone ameliorated colitis and suppressed pro-inflammatory gene expression. In conclusion, our data indicates that miRNA-146b and PPARγ activation may be implicated in the regulation of Th17 responses and colitis in C. difficile-infected mice. Click here to read the article. SUMMARY Clostridium difficile infection in WT mice resulted in a strong inflammatory immune response characterized by colonic leukocyte infiltration and mucosal thickening, as well as an increased production of pro-inflammatory cytokines including MCP-1, IL-6, IL-17 and IL-1β. miRNA-sequencing results revealed an up-regulation of three microRNAs within infection: miR-146b, miR-1940 and miR-1298 (Figure 1). MiRNAs are small (∼22–24-nucleotide), non-coding, single-stranded RNA molecules that contribute to regulating gene expression. Mature miRNAs are incorporated into the RNA-induced silencing complex where they typically recognize and bind to sequences in the 3′ untranslated regions, leading to suppression of translation and/or degradation of mRNA.

Modeling the role of peroxisome proliferator-activated receptor γ and microRNA-146 in mucosal immune responses to Clostridium difficile.

Clostridium difficile infection is currently the leading cause of diarrhea in the healthcare setting, resulting in an average of 14,000 deaths in the US each year. The incidence and severity of C. difficile infection is increasing, due in part to the emergence of new strains that present higher sporulation, toxin production and resistance to antibiotics. Current available treatments are unsuccessful in bacterial clearance and complete patient remission as illustrated by the 30% disease relapse in infected individuals.

Clostridium difficile is an environmental Gram-positive, anaerobic, spore-forming bacillus belonging to the phylum Firmicutes. It is currently the leading cause of diarrhea in the healthcare setting and becoming an increasingly common cause of diarrhea in industrialized countries [1]. Although it is not considered part of the human microbiome, C. difficile colonizes and can be isolated from about 1-5% of healthy adult population and 75% of infant mammals, with an increase to 39% in hospitalized patients [2]. However, only the toxin-producing strains are associated with the diarrheic disease C. difficile infection (CDI). Many countries have reported an increase in both incidence and severity of CDI over the last few years [3-5], probably due to the emergence of hypervirulent strains including BI/NAP1/027. These new quinolone-resistant, hypertoxinogenic, binary toxin-producing C. difficile strains pose a growing threat to most health care settings, where patients accidentally ingest spores of the bacterium in the context of destruction of commensal bacteria due to broad-spectrum antibiotic treatment. Therefore, such recently emerged C. difficile strains are responsible for increased morbidity, mortality and healthcare costs in the US, Canada and Europe, primarily due to toxic megacolon, septic shock, intestinal perforation, and resistance to fluoroquinolone antibiotics [2, 6-8]. Some examples of recent relevant outbreaks include:

• Quebec, 2002: 17% mortality [9]
• Quebec, 2003: 13.4% mortality [9]
• 2000-2010, Montreal: Estimate of 2,000 total deaths
• 2003, St. Louis: 6% mortality [10, 11]

According to the Agency for Healthcare Research and Quality Nationwide Inpatient Sample, there were approximately 139,000 patients discharged from acute care facilities who were diagnosed with C. difficile infection in 2000. This number increased to 336,000 patients in 2008. In the United States alone, CDI was estimated to affect over 500,000 people and to cost the healthcare system over $3 billion in 2009 [12].

CDI is a nosocomial toxin-mediated intestinal disease which can result in a wide range of clinical outcomes:

• Asymptomatic colonization
• Mild to severe diarrhea
• Abdominal pain
• Fever
• Leukocytosis
• Colitis with or without the presence of pseudomembranes
• Toxic megacolon or bowel perforation
• Sepsis
• Death

Other rare manifestations associated with CDI are:

• Extra-intestinal infections
• Ileal infections
• Reactive arthritis
• Post-proctocolectomy enteritis
• Bacteremia

The severity of the symptoms mostly depends on the properties of the C. difficile strain, which is usually characterized by the genetic-dependent ability to produce toxins.

Prevention and control of C. difficile infection is a major public health challenge. Advanced age is a risk factor for development and death from C. difficile-associated disease. In addition, the Centers for Disease Control and Prevention have reported expanded susceptible populations that now include children and previously healthy young women. While antibiotics are effective in inhibiting the organism and treating symptoms, recurrent C. difficile infection occurs in up to 15-30 % of cases after cessation of treatment. Management of recurrent C. difficile infection has met with variable success and too often failure. Thus, a better understanding of mucosal immune responses to C. difficile is needed for the rational development of novel therapies against this organism. Several studies have been published related to immune responses to C. difficile: 1. C. difficile toxins TcdA and TcdB breach the intestinal barrier and trigger mucosal inflammation and intestinal damage. The inflammasome is an intracellular danger sensor of the innate immune system. This study found that TcdA and TcdB triggered inflammasome activation and IL-1β secretion in macrophages and in human mucosal biopsy specimens. The IL-1β release required recognition of the full-length toxin but not its enzymatic function. Pre-treating mice with the IL-1 receptor antagonist anakinra protected the host to a degree similar to that seen in the caspase 1 adaptor ASC^-/- mice. These data suggest new therapeutic targets in the treatment of C. difficile-associated disease [13]. 2. Fidaxomicin is a new antibiotic with increased in vitro activity again C. difficile and a low rate of absorption from the intestinal tract. This phase 3 clinical trial compared the efficacy and safety of fidaxomicin with those of vancomycin (standard therapy) in treating C. difficile infection. A total of 629 patients were randomized to receive one drug or the other orally for 10 days. Rates of initial symptomatic relief were similar for both drugs (intention-to-treat analysis, 88.2% versus 85.8%). However, significantly fewer patients in the fidaxomicin group than in the vancomycin group has a recurrence of the infection (intention-to-treat analysis, 15.4% versus 25.3%). The adverse-event profile was similar for the two therapies. These results suggest a new antibiotic for front-line therapy of C. difficile infection [14]. 3. New therapies are needed to combat the high rate of recurrence of C. difficile infection. A randomized, double-blind placebo-controlled study has been recently reported of two neutralizing, fully human monoclonal antibodies against C. difficile toxins A (CDA1) and B (CDB1). Antibodies were administered as a single IV infusion, 10 mg/Kg of body weight, in patients with symptomatic C. difficile infection receiving metronidazole or vancomycin. Among 200 patients enrolled, the rate of recurrence of C. difficile infection was significantly lower among those treated with monoclonal antibodies (7% vs. 25%, p<0.001). Among patients with more than one previous episode of C. difficile infection, recurrence rates were 7% and 38%, respectively (p<0.006). The serious adverse event rate was not different between the two groups. This study suggests that the addition of monoclonal antibodies against

C. difficile toxins to antibiotic agents significantly reduces the recurrence of C. difficile infection [15]. In light of the unsuccessful treatments that have been discovered and developed so far, MIEP is developing non-antibiotic (pharmacological) means of reducing the acute level of intestinal inflammation and preventing disease recurrence; using computational modeling approaches to identify pro-inflammatory mucosal cell types and/or locally generated cytokines that could be targeted to reduce clinical disease, yet allowing normal commensal microorganisms to re-populate the intestinal lumen; and developing an immuno-diagnostic that identifies early-on patient cases likely to have a severe course of disease (including recurrence) who will then be given alternative therapies.

Previous studies have demonstrated that three days of pre-treatment with a mixture of antibiotics in the drinking water for 3 days is sufficient to disrupt the intestinal microflora and allow C. difficile infection [16]. The antibiotic mixture consists of colistin 850 U/mL (which corresponds to 4.2 mg/kg), gentamicin 0.035 mg/mL (which corresponds to 3.5 mg/kg), metronidazole 0.215 mg/mL (which corresponds to 21.5 mg/kg) and vancomycin 0.045 mg/mL (which corresponds to 4.5 mg/kg). A control group receiving no antibiotics is also included. Following the treatment all mice are given regular autoclaved water for 2 days and all mice including the control group receive a single dose of clindamycin (32 mg/kg) intraperitoneally 1 day before C. difficile challenge. On day 5 of the study mice are infected intragastrically with Clostridium difficile (10⁷ cfu of strain VPI 10463 in the MIEP studies) (Figure 1). Mice are then weighed and scored daily to assess mortality and morbidity and the presence of diarrhea and other symptoms.

Under the MIEP program we have investigated the role of Peroxisome Proliferator-Activated Receptor g and MicroRNA-146 in Mucosal Immune Responses to Clostridium difficile [17]. Specifically, we have applied computational and mathematical modeling approaches in combination with RNA-sequencing and mouse challenge studies to characterize the disruption of an important regulatory pathway during Clostridium difficile infection. We have found that tissue damage and disease severity in C. difficile infection is associated with a disruption of the peroxisome proliferator-activated receptor gamma (PPARγ) pathway, including a possible relationship between this nuclear receptor and microRNAs (Figure 2). Activation of PPARγ by using a diabetes drug ameliorated C. difficile-related gut pathology and disease in mouse challenge studies. Thus, PPARγ helps keep the immune response in check, allowing the body to heal but also allowing the immune cells that fight infection to do their work in a controlled manner. When PPARγ was absent or not activated, disease was more rampant and colonic lesions from C. difficile were much worse. For more information regarding this project, click here.

1. Lo Vecchio, A. and G.M. Zacur, Clostridium difficile infection: an update on epidemiology, risk factors, and therapeutic options. Curr Opin Gastroenterol, 2012. 28(1): p. 1-9.

2. Barbut, F., G. Jones, and C. Eckert, Epidemiology and control of Clostridium difficile infections in healthcare settings: an update. Curr Opin Infect Dis, 2011. 24(4): p. 370-6.

3. Lim, P.L., et al., Increasing incidence of Clostridium difficile-associated disease, Singapore. Emerg Infect Dis, 2008. 14(9): p. 1487-9.

4. Khanna, S. and D.S. Pardi, The growing incidence and severity of Clostridium difficile infection in inpatient and outpatient settings. Expert Rev Gastroenterol Hepatol, 2010. 4(4): p. 409-16.

5. Zilberberg, M.D., A.F. Shorr, and M.H. Kollef, Increase in adult Clostridium difficile-related hospitalizations and case-fatality rate, United States, 2000-2005. Emerg Infect Dis, 2008. 14(6): p. 929-31.

6. Loo, V.G., et al., A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality. N Engl J Med, 2005. 353(23): p. 2442-9.

7. Khanna, S., et al., The epidemiology of community-acquired Clostridium difficile infection: a population-based study. Am J Gastroenterol, 2012. 107(1): p. 89-95.

8. Kuehn, B.M., Scientists seek strategies to prevent Clostridium difficile infections. JAMA, 2011. 306(17): p. 1849-50.

9. Weiss, K., et al., Multipronged intervention strategy to control an outbreak of Clostridium difficile infection (CDI) and its impact on the rates of CDI from 2002 to 2007. Infect Control Hosp Epidemiol, 2009. 30(2): p. 156-62.

10. Dubberke, E.R., et al., Attributable outcomes of endemic Clostridium difficile-associated disease in nonsurgical patients. Emerg Infect Dis, 2008. 14(7): p. 1031-8.

11. Warny, M., et al., Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe. Lancet, 2005. 366(9491): p. 1079-84.

12. Rupnik, M., M.H. Wilcox, and D.N. Gerding, Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat Rev Microbiol, 2009. 7(7): p. 526-36.

13. Ng, J., et al., Clostridium difficile toxin-induced inflammation and intestinal injury are mediated by the inflammasome. Gastroenterology, 2010. 139(2): p. 542-52, 552 e1-3.

14. Louie, T.J., et al., Fidaxomicin versus vancomycin for Clostridium difficile infection. N Engl J Med, 2011. 364(5): p. 422-31.

15. Lowy, I., et al., Treatment with monoclonal antibodies against Clostridium difficile toxins. N Engl J Med, 2010. 362(3): p. 197-205.

16. Chen, X., et al., A mouse model of Clostridium difficile-associated disease. Gastroenterology, 2008. 135(6): p. 1984-92.

17. Viladomiu, M., et al., Modeling the role of peroxisome proliferator-activated receptor gamma and microRNA-146 in mucosal immune responses to Clostridium difficile. PLoS One, 2012. 7(10): p. e47525.

Enteroaggregative E. coli (EAEC) is an etiologic agent of enterocolitis and severe persistent diarrhea worldwide, especially in malnourished children and immunosuppressed individuals. While many E. coli strains co-exist in the gastrointestinal tract as harmless commensal symbionts, disease-causing pathogenic E. coli strains have the ability to induce life-threatening illnesses that often require hospitalization and can result in death. EAEC is one of six pathotypes known to induce intestinal disease and persistent diarrhea. Epidemiologic studies reveal EAEC as the single most common cause of bacteria-induced diarrhea among all age groups in the United States [1]. EAEC also ranks as the second most common cause of traveler’s diarrhea [2]. Both developing and industrialized countries are susceptible to experiencing large EAEC-induced diarrheal outbreaks presenting significant public health concerns [3]. The recent German outbreak in May 2011 caused by shiga toxin producing EAEC strain O104:H4 resulted in the highest frequency of EAEC-induced deaths ever recorded [4].

EAEC pathogenesis has been summarized into three primary stages:

1. Abundant adherence to the intestinal mucosa
2. Biofilm formation over enterocyte surface
3. Release of toxins, induction of proinflammatory response, mucosal toxicity, and enhanced intestinal secretion [3,5]

Several well defined virulence factors exist in the EAEC genome however the complete understanding of pathogenesis remains unknown. A few virulence factors are briefly described below:

• Aggregative adherence fimbria (AAF) mediate the adhesion of EAEC to the intestinal mucosa favoring the formation of the thick biofilm. At least four distinct AAF alleles have been described and are known to contribute to the inflammatory response of epithelial cells infected with EAEC [6].
• Dispersin is an EAEC-secreted protein necessary for the proper extension of AAF [7].
• Pic is a serine autotransporter with mucinolytic properties allowing EAEC to penetrate the mucus layer of the intestine and induce mucin hypersecretion [8].
• Pet protein is an enterotoxin that causes severe damage to epithelial cells by cleaving cytoskeleton proteins within the microvilli [9].
• Heat-stable enterotoxin-1 (EAST1) secreted by EAEC may promote the initial phase of watery diarrhea by enhancing intracellular levels of cGMP and inhibiting the Na/Cl transport system [10].

The exceptionally virulent shiga toxin-producing EAEC strain O104:H4 illuminates how plasmid-encoded virulence factors can be gained suggesting that that the EAEC archetype is not as fixed as once believed [11].

EAEC flagellin are known to mediate the activation of inflammatory pathways in epithelial cells through toll-like receptor signaling resulting in proinflammatory cytokine production. IL-8 is a hallmark cytokine produced by epithelial cells during EAEC infection that promotes neutrophil recruitment and transmigration [12]. Recently the MIEP team has discovered the importance of Th17 cells in host responses to EAEC facilitating bacterial clearance. We modulated mucosal inflammation through pharmacological and genetic ablation of transcription factor PPARg in malnourished hosts. Loss of PPARg functionality promoted a beneficial proinflammatory response generating a cytokine environment prone to Th17 cell differentiation and recruitment that lead to disease amelioration. Cellular responses in EAEC-infected PPARg deficient mice were accompanied by enhanced antimicrobial peptide production likely mediated by an IL-17 dependent mechanism. Additional studies are geared toward understanding how EAEC induces these responses.

A high demand exists for a reproducible and comprehensive animal model of EAEC infection to elucidate novel mechanisms of host-EAEC interactions. Currently, four primary animal models exit:

1. Gnotobiotic piglet model [13]
2. Neonatal and weaned C57BL/6 mice with or without malnutrition [14]
3. Antibiotic treatment in combination with infection in adult BALB/c mice [15]
4. Immunodeficient mice with human intestinal xenograph implantation [16]

These models have provided insight related to EAEC pathogenesis however they are highly limited in their ability to characterize immunological responses to EAEC at the intestinal mucosa. There is still a desperate need for the development of an animal model that reproduces significant symptoms of EAEC-induced pathology.

MIEP specifically focuses on characterizing the mechanisms underlying EAEC infection by integrating computational and mathematical modeling approaches with mouse challenge studies to identify novel biomarkers and discover unforeseen therapeutic targets. Ultimately, MIEP projects aim to develop modeling aided, broad-based, host-targeted immune therapeutics that will protect against EAEC-induced mucosal damage and persistent diarrhea. Currently our projects focus on prototype strains 042 and JM221. In October 2012 we released the first version of the EAEC T cell differentiation model. This model is an oversimplified depiction of EAEC infection, antigen presentation, and the host’s adaptive immune response to pathogen. We calibrated the model using in house gene expression and flow cytometry data from malnourished EAEC strain JM221 infected mice. The goal of the simplified EAEC model is to capture and quantify the dynamics of interest surrounding EAEC-host interaction; specifically T cell responses and their contributory effects on bacterial clearance. Currently the model effectively predicts the beneficial role of an early proinflammatory and Th17 response in disease amelioration through EAEC clearance by day 5. Additionally, the model is successful in matching the assay-based measurements from in vivo experiments during simulations. As more data is generated through validation studies, the model will be refined and built up to encapsulate additional cell types and more complex molecular pathways. In doing so, we aim to predict non-intuitive physics underlying host-EAEC interactions in a holistic manner.

1. Nataro JP, Mai V, Johnson J, Blackwelder WC, Heimer R, et al. (2006) Diarrheagenic Escherichia coli infection in Baltimore, Maryland, and New Haven, Connecticut. Clin Infect Dis 43: 402-407.

2. Taylor DN, Bourgeois AL, Ericsson CD, Steffen R, Jiang ZD, et al. (2006) A randomized, double-blind, multicenter study of rifaximin compared with placebo and with ciprofloxacin in the treatment of travelers’ diarrhea. Am J Trop Med Hyg 74: 1060-1066.

3. Huang DB, Mohanty A, DuPont HL, Okhuysen PC, Chiang T (2006) A review of an emerging enteric pathogen: enteroaggregative Escherichia coli. J Med Microbiol 55: 1303-1311.

4. Al Safadi R, Abu-Ali GS, Sloup RE, Rudrik JT, Waters CM, et al. (2012) Correlation between in vivo biofilm formation and virulence gene expression in Escherichia coli O104:H4. PLoS One 7: e41628.

5. Nataro JP (2005) Enteroaggregative Escherichia coli pathogenesis. Curr Opin Gastroenterol 21: 4-8.

6. Harrington SM, Strauman MC, Abe CM, Nataro JP (2005) Aggregative adherence fimbriae contribute to the inflammatory response of epithelial cells infected with enteroaggregative Escherichia coli. Cell Microbiol 7: 1565-1578.

7. Sheikh J, Czeczulin JR, Harrington S, Hicks S, Henderson IR, et al. (2002) A novel dispersin protein in enteroaggregative Escherichia coli. J Clin Invest 110: 1329-1337.

8. Navarro-Garcia F, Gutierrez-Jimenez J, Garcia-Tovar C, Castro LA, Salazar-Gonzalez H, et al. (2010) Pic, an autotransporter protein secreted by different pathogens in the Enterobacteriaceae family, is a potent mucus secretagogue. Infect Immun 78: 4101-4109.

9. Canizalez-Roman A, Navarro-Garcia F (2003) Fodrin CaM-binding domain cleavage by Pet from enteroaggregative Escherichia coli leads to actin cytoskeletal disruption. Mol Microbiol 48: 947-958.

10. Telli M, Guiral E, Martinez JA, Almela M, Bosch J, et al. (2010) Prevalence of enterotoxins among Escherichia coli isolates causing bacteraemia. FEMS Microbiol Lett 306: 117-121.

11. Mellmann A, Harmsen D, Cummings CA, Zentz EB, Leopold SR, et al. (2011) Prospective genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology. PLoS One 6: e22751.

12. Khan MA, Kang J, Steiner TS (2004) Enteroaggregative Escherichia coli flagellin-induced interleukin-8 secretion requires Toll-like receptor 5-dependent p38 MAP kinase activation. Immunology 112: 651-660.

13. Tzipori S, Montanaro J, Robins-Browne RM, Vial P, Gibson R, et al. (1992) Studies with enteroaggregative Escherichia coli in the gnotobiotic piglet gastroenteritis model. Infect Immun 60: 5302-5306.

14. Roche JK, Cabel A, Sevilleja J, Nataro J, Guerrant RL (2010) Enteroaggregative Escherichia coli (EAEC) impairs growth while malnutrition worsens EAEC infection: a novel murine model of the infection malnutrition cycle. J Infect Dis 202: 506-514.

15. Harrington SM, Sheikh J, Henderson IR, Ruiz-Perez F, Cohen PS, et al. (2009) The Pic protease of enteroaggregative Escherichia coli promotes intestinal colonization and growth in the presence of mucin. Infect Immun 77: 2465-2473.

16. Boll EJ, Struve C, Sander A, Demma Z, Nataro JP, et al. (2012) The Fimbriae of Enteroaggregative Escherichia coli Induce Epithelial Inflammation In Vitro and in a Human Intestinal Xenograft Model. J Infect Dis 206: 714-722.