January/February 2017

Harnessing the Immune System to Treat C diff Infection
By Carrie A. Cowardin, PhD
Today's Geriatric Medicine
Vol. 10 No. 1 P. 28

Clostridium difficile (C diff) is a bacterial pathogen that was first identified as the cause of antibiotic-associated diarrhea in the late 1970s.1 Today, C diff causes an estimated 500,000 infections per year and has become the most common hospital-acquired infection in the United States.2 Although community-acquired cases are increasingly common, hospitals and long term care facilities are particularly vulnerable to the spread of C diff, which forms hardy chemical- and oxygen-resistant spores. Patients typically become infected with C diff following antibiotic treatment, which disrupts the healthy bacteria found in the gut that are responsible for out-competing the pathogen and promoting beneficial immune responses. Additional risk factors include age, inflammatory bowel disease, immunosuppression, and use of proton-pump inhibitors.3

C diff spores are transmitted via the fecal-oral route and germinate into actively dividing bacteria upon reaching the intestine. This pathogen produces toxins that are primarily responsible for the symptoms of infection, as nontoxigenic strains are not associated with disease. Symptoms of C diff infection (CDI) range from mild diarrhea to life-threatening pseudomembranous colitis and toxic megacolon. Treatment involves stopping the offending antibiotic that preceded infection and administration of metronidazole or vancomycin, both of which kill C diff. Currently, trials are under way to test fecal microbiota transplantation as a treatment for CDI. Preliminary evidence suggests this approach to restore healthy gut microbiota is effective; however, it is not without risks, as our current understanding of the role of the microbiota in diverse health conditions is limited.4

One of the major challenges to successful CDI management is frequent disease recurrence, as 10% to 35% of infected patients become reinfected following treatment. This is likely complicated by the fact that the very antibiotics used to treat disease may also perpetuate the disruption of healthy gut bacteria. Reinfection frequently involves a second strain of C diff, suggesting that the original treatment effectively eradicates the pathogen but patients remain susceptible to new infection.5 Successful resolution of CDI involves generation of a protective immune response against the bacterium and its toxins, and recurrence has been associated with significantly lower levels of antitoxin antibodies.6 Indeed, the type and intensity of inflammation generated in response to C diff appear to be a crucial determinant of outcome, as recent studies show high levels of the inflammatory cytokines interleukin-8 and chemokine ligand 5 were better predictors of severe disease than C diff burden.7,8

"Hypervirulent" C diff
Many different strains of C diff with a variety of distinct characteristics have been identified. These strains are frequently designated according to their polymerase chain reaction ribotype, a technique that involves generating specific signatures for differing strains via digestion and visualization of ribosomal DNA. Perhaps the best known ribotype of C diff is the infamous ribotype 027. Strains belonging to this classification caused severe outbreaks of CDI in the United States and the United Kingdom, and have since spread worldwide.

Ribotype 027 strains demonstrate increased antibiotic resistance, most notably to the fluoroquinolone class of antibiotics, and multiple studies have demonstrated an association between infection with ribotype 027 strains and highly transmissible severe disease with increased rates of recurrence.9-11 However, other studies have failed to find associations between ribotype 027 strains and more severe disease, prompting debate within the field over the classification of the strains as hypervirulent. In fact, the conflicting findings may implicate differences between individual ribotype 027 strains, the environment in which the infection occurs, or other unidentified patient characteristics.

The Binary Toxin C diff Transferase
Toxins A and B, the major toxins produced by C diff, are capable of killing human cells and inducing inflammation within the colon. However, certain strains of C diff also produce an additional toxin known as C diff transferase (CDT). This toxin is also sometimes referred to as "binary toxin" or "Toxin C," due both to its two components and its more recent designation. Strains that produce this toxin include but are not limited to the ribotype 027 strains, as well as ribotype 078 isolates, which have recently been found in both pigs and humans.12

CDT-positive strains have become increasingly common over the last 15 years, stimulating investigation into the role of this toxin during disease. Evidence exists to show that CDT could enhance adherence of C diff to human cells, suggesting that it may promote the ability of C diff to infect patients.13 However, it was unclear whether or how this toxin could impact the immune response of infected individuals. Because the generation of a healthy immune response to C diff is critical to infection outcome, determining how CDT influences the course of disease and the immune response to infection is critical to understanding its role in disease.

Eosinophils and a Protective Immune Response to CDI
Our recent findings suggest that CDT does indeed contribute to the pathogenesis of CDI, as mutation of the toxin in two distinct ribotype 027 strains decreased the severity of infection in a mouse model of disease. Interestingly, deletion of the toxin did not influence the infectious burden of C diff but did result in changes in the host immune response during infection. When CDT was produced, infection resulted in a reduction in the number of host eosinophils, while other cell types remained unchanged. Eosinophils, although classically associated with immune responses to allergy or helminth infection, were found to be important for survival during infection with C diff. Depletion of eosinophils resulted in more severe infection even in the absence of CDT, and restoring eosinophils that could not recognize the toxin protected mice during infection.14

These results agree with previous findings demonstrating the protective capacity of eosinophils during CDI. Indeed, eosinophils appear to be controlled at least in part by the microbiota-regulated signaling molecule interleukin-25, which is able to boost eosinophil responses and protect mice from death due to infection.15 Although it is unclear what beneficial function these cells perform, their capacity to promote tissue healing and antibody production is well known in other contexts and seems to be a promising target.16

New Perspective on Inflammation
These findings herald a shift in the way inflammation is conceptualized in response to C diff. It is now clear that certain types of inflammation are more beneficial than others during this disease, with an eosinophil-centered response being particularly protective. In contrast, inflammation dominated by the cytokine interleukin-23 appears to be pathogenic, as mice deficient for this cytokine show improved survival during infection. Interestingly, interleukin-23 promotes a neutrophil-centered response and appears to be reciprocally regulated with interleukin-25, which promotes eosinophils. These results suggest that not all inflammation is "good" during CDI but reinforce the idea that the type of immune response generated plays a major role in determining a positive or negative outcome.

This view of infection gives hope to the idea of directing the immune system to treat acute CDI or to prevent disease recurrence in a more targeted fashion, and suggests that including CDT as a target for future vaccines or antibody-based therapies is essential. Although more work is required to demonstrate the feasibility of these approaches, particularly in human studies, these results open promising areas for the development of new treatments for CDI.

— Carrie A. Cowardin, PhD, who completed her doctoral work on the immune response to C diff in the laboratory of William A. Petri, Jr, MD, PhD, at the University of Virginia, is a postdoctoral research associate at Washington University in St. Louis, focused on the communication between gut microbes and the host immune and skeletal systems.

References
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13. Schwan C, Stecher B, Tzivelekidis T, et al. Clostridium difficile toxin CDT induces formation of microtubule-based protrusions and increases adherence of bacteria. PLoS Pathog. 2009;5(10):e1000626.

14. Cowardin CA, Buonomo EL, Saleh MM, et al. The binary toxin CDT enhances Clostridium difficile virulence by suppressing protective colonic eosinophilia. Nat Microbiol. 2016;1(8):16108.

15. Buonomo EL, Cowardin CA, Wilson MG, Saleh MM, Pramoonjago P, Petri WA. Microbiota-regulated IL-25 increases eosinophil number to provide protection during Clostridium difficile infection. Cell Rep. 2016;16(2):432-443.

16. Travers J, Rothenberg ME. Eosinophils in mucosal immune responses. Mucosal Immunol. 2015;8(3):464-475.