The NIMML combines experimental and computational methods to study the mechanisms of action underlying immune responses to Helicobacter pylori, Escherichia coli, Clostridium difficile, and influenza virus. Infectious diseases are caused by pathogenic microorganisms, such as bacteria, viruses, parasites or fungi; the diseases can be spread, directly or indirectly, from one person to another.

Modulation of hepatic PPAR expression during Ft LVS LPS-induced protection from Francisella tularensis LVS infection.

Novel insights on the role of CD8+ T cells and cytotoxic responses during Helicobacter pylori infection.

Predictive computational modeling of the mucosal immune responses during Helicobacter pylori infection.

Helicobacter pylori infection in a pig model is dominated by Th1 and cytotoxic CD8+ T cell responses.

Mutations to essential orphan response regulator HP1043 of Helicobacter pylori result in growth-stage regulatory defects.

Helicobacter pylori colonization ameliorates glucose homeostasis in mice through a PPAR γ-dependent mechanism.

Host-interactive genes in Amerindian Helicobacter pylori diverge from their Old World homologs and mediate inflammatory responses.

Animal models of enteroaggregative Escherichia coli infection.

Enteroaggregative Escherichia coli strain in a novel weaned mouse model: exacerbation by malnutrition, biofilm as a virulence factor and treatment by nitazoxanide.

The role of peroxisome proliferator-activated receptor γ in immune responses to enteroaggregative Escherichia coli infection.

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

Lanthionine synthetase component C-like protein 2: a new drug target for inflammatory diseases and diabetes.

Dietary abscisic acid ameliorates influenza-virus-associated disease and pulmonary immunopathology through a PPARγ-dependent mechanism.

PPAR-gamma activation as an anti-inflammatory therapy for respiratory virus infections.

Arachidonic acid-and docosahexaenoic acid-enriched formulas modulate antigen-specific T cell responses to influenza virus in neonatal piglets.

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 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.

Link to the raw data can be found here.

Link to the publication can be found here.

Link to the press release can be found here.

1. Helicobacter pylori infection in a pig model is dominated by Th1 and cytotoxic CD8+ T cell responses. [PubMed]
2. Novel insights on the role of CD8+ T cells and cytotoxic responses during Helicobacter pylori infection. [PubMed]

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.

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.

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.

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