The Inflammasome and Type-2 Immunity in Clostridium difficile Infection

Abstract

Clostridium difficile (reclassified as “ Clostridioides ”) is the leading cause of hospital-acquired infections in the United States, and is associated with high-patient mortality and high rates of recurrence. Inflammasome priming and activation by the bacterial toxins, TcdA , TcdB , and C. difficile transferase (CDT), initiates a potent immune response that is characterized by interleukin- (IL) 8, IL-1β, and neutrophil recruitment, and is required for pathogen killing. However, it is becoming clearer that a strong inflammatory response during C. difficile infection can result in host tissue damage, and is associated with worse patient outcome. Recent work has begun to show that a type-2 immune response, most often associated with helminth infections, allergy, and asthma, may be protective during C. difficile infection. While the mechanisms through how this response protect are still unclear, there is evidence that it is mediated through eosinophil activity. This chapter will review the immune response to C. difficile, how the inflammasome signaling during infection can deleterious to the host, as well as the current understanding of a protective type-2 immunity. Understanding the host immune response may help to provide insight into novel approaches to prognosis markers, as well as how treat patient C. difficile infection without, or in addition to, antibiotics.

Clostridium difficile , ^a the gram-positive, spore-forming, and obligate anaerobe is the leading hospital-acquired infection in the United States, resulting in mild to severe diarrhea, pseudomembranous colitis, toxic megacolon, and even death. C. difficile infection (CDI) is most often associated with prior disruption to the normal colon microbiota as a result of antibiotic usage which allows for a competitive advantage for C. difficile growth. In patients white blood cell (WBC) count above 20 × 109/L and high-inflammatory levels are indicators of severe disease, suggesting that the host response to infection may play some role in the outcome of patient morbidity and mortality. ^{1 2}

The two main virulence factors of C. difficile are toxins A and B that can lead to cell death and result in the characteristic epithelial damage associated with CDI. ³ These toxins have been observed to activate the inflammasome, leading to a robust inflammatory response that, when in abundance, can be pathogenic and increase host morbidity and mortality. ⁴ A more recently characterized toxin, now found in upwards of 20% of clinical isolates, ^{4 5 6} is the binary toxin C. difficile transferase (CDT). The presence of this toxin is associated with increased severity and mortality in patients, ^{7 8} but the mechanism through which this occurs during disease has only recently been elucidated, and appears to involve toll-like receptor-2 (TLR2) signaling. ⁹

Infection with C. difficile is acute, is characterized by a high-inflammatory profile, and the innate immune response is thought of as being highly critical. ¹⁰ Recently, it has been shown that eosinophils are also important for protection from CDI ^{9 11 12} and may work to repair tissue damage caused by neutrophil activity, though the mechanisms of action remain unknown. Interestingly, eosinophils are a characteristic immune cell of type-2 immunity, a response which is most often associated with helminth infections, allergy, and asthma. ^{13 14 15} This chapter will explore the immune response to C. difficile infection, focusing mainly on the inflammasome activation by bacterial toxins and the role of type-2 immune responses within the host.

Type-2 Immune Response

Infection with C. difficile is often characterized by high levels of proinflammatory cytokines, such as interleukin- (IL) 1β, IL-8, IL-23, and IFNγ (interferon-gamma), as well as the high recruitment of neutrophils. ^{2 16} The release of IL-1β and IL-8 is dependent upon pathogen-associated inflammasome activation ¹⁷ which is an important step in alerting a proinfammatory response during C. difficile , ⁴ including neutrophil recruitment. Comprising a large proportion of the WBC population, neutrophil recruitment to the colon of patients during acute infection, is essential for host protection through direct killing of the bacteria through phagocytosis and release of microbicidal factors. ¹⁸ Their importance is highlighted by the observation that mice that lack effective neutrophil responses have increased disease severity and mortality ¹⁹ indicating that an effective immune response through neutrophils is important in C. difficile infection. However, when found in overwhelming numbers, neutrophils can be harmful by inducing high levels of tissue damage and can correlate with increased mortality, ^{3 18} highlighting the importance of a balanced immune response.

In contrast, a type-2 immune response generally refers to a profile of Th2 cytokines, such as IL-4, IL-5, and IL-13, as well as production of antibodies from plasma cells, particularly members of the immunoglobulin (Ig) E subclass. ²⁰ Additionally, it employs the function of immune cells, such as “alternatively-activated” macrophages, eosinophils, Th2 CD4 ⁺ cells, and B cells. It is most often induced during infection with parasites, such as helminths to facilitate pathogen killing and expulsion from host, while attempting to limit host damage and promote tissue repair. ^{20 21} When induced inappropriately, type-2 immunity is thought to be the driving factor behind allergic and asthmatic responses. ^{15 22} In these cases, the immune response is induced by nonpathogenic foreign particles and promotes pathogenic tissue inflammation and damage, often resulting in long-term tissue remodeling. ²³

During C. difficile infection, the strong proinflammatory response a characteristic of infection, and often can be used clinically to assess severity and predict patient outcome. However, the presence of a type-2 immune response during CDI is a novel finding, and in this chapter the current evidence supporting how this is protective will be discussed.

Antibiotics, IL-25, and C. difficile Infection

The most serious and common risk factor for contraction of C. difficile infection is the use of antibiotics, which disrupt the normal microbiota of the colon, and may allow for competitive advantages for C. difficile. ¹⁶ The ability of the normal microbiome to outcompete pathogens for space and nutrients is called colonization resistance that is thought to be the primary way that humans protect against CDI in the absence of antibiotic usage. Colonization resistance of the normal microbiota may be, in part, attributed to certain byproducts of resident commensal bacteria, including secondary bile acids, such as deoxycholate. ²⁴ These bile acids may suppress ability of C. difficile spores to germinate within the colon and prevent growth. However, with use of antibiotics, secretion of the secondary bile acid decreases, while primary bile acids, such as taurocholate, increase. In contrast to secondary bile acids, taurocholate promotes germination of spores and vegetative cell expansion. ^{25 26} Additionally, Buonomo et al, showed that antibiotic usage contributes to a large decrease in IL-25 levels within the colon.

IL-25, an important type-2 cytokine, is produced in the epithelium of the intestine by the Tuft cells, ²⁷ as Th2 CD4+ cells and mast cells, among others; it can be considered a first-responding cytokine, as it works to kickstart a type-2 inflammatory response. ²⁸ Downstream, it leads to an increase in type 2 cytokines and immune cells, such as IL-4, IL-5, eosinophils, and macrophages, which work to limit and clear infections or promote allergic or asthmatic responses. ^{28 29} Additionally, IL-25 has been seen to negatively correlate with IL-23 levels, ³⁰ which may suggest that this pathway works opposing to IL-23 signaling through either direct or indirect regulation of the proinflammatory cytokine.

Buonoma et al observed that mice given antibiotics were observed to have decreased levels of colon tissue IL-25, and this decrease continued throughout infection with C. difficile . Additionally, when supplemented with recombinant IL-25, mice were significantly protected from CDI related mortality and morbidity, and has improved tissue damage when observed by histology. This suggests that IL-25 mediates protection, and this is abrogated following antibiotic usage. Further, the addition of IL-25 increased colonic eosinophil levels, and this was shown to be the mechanism by which IL-25 provided protection. The role of eosinophils during infection will be discussed later. Buonomo et al, also compared human histology sections from patients with and without CDI-associated diarrhea, and observed that CDI patients had significantly lower levels of IL-25, supporting the conclusion that this cytokine is involved during antibiotic usage and infection, and that its role may be protective. Taken together, these results suggest that the microbiota regulation of IL-25 levels is an important mediator in pathogenesis and disease severity of C. difficile infection and that antibiotics promote susceptibility through multiple mechanisms, including reducing colonization resistance and immune modulation.

C. difficile Activates the Inflammasome

C. difficile 's main virulence factors, toxins A and B, are responsible for glycosylation of the Rho family of GTPases which causes the loss of activity of RhoA, Rac1, and Cdc42, and leads to epithelial cell death. ³¹ Additionally, these toxins are sensed by host cells through both mitogen-activated protein (MAP) kinases and the pyrin inflammasome. ^{32 33} Through this, toxins A and B can stimulate a potent proinflammatory response through IL-1β, and IL-8, tumor necrosis factor α (TNF-α), and IL-6. ^{34 35}

Activation of the inflammasome is an important mediator in innate immune responses and can lead to early recruitment of immune cells that are important for host survival. A two-step process, inflammasome activity requires both a priming and an activating step before cleaving and leading to pyroptosis and release of proinflammatory cytokines. ^{36 37} Pattern recognition receptor (PRR) engagement with pathogen-associated molecular patterns (PAMPs) or bacterial toxins are thought to be the canonical priming step for inflammasomes. ³⁶ Once primed, in the context of C. difficile infection, the pyrin inflammasome can be activated through sensing the inactivity of the Rho family GTPases due to toxins A and B. ³⁵ This leads to cleaving of pro-IL-1β and IL-8 those are released from the cells from pores formed in the plasma membrane. IL-1β and IL-8 are potent proinflammatory cytokines and can result in the recruitment of innate immune cells that are important for pathogen killing. ^{36 37} Therefore, the presence of inflammasome signaling can provide an early and important mediator in immune protection from pathogens. Conversely, Cowardin et al showed that robust IL-1β signaling leads to the induction of the cytokine IL-23 which is correlated with increased severity of disease in CDI.

IL-23, a potent proinflammatory cytokine, is induced downstream of TLR and IL-1R signaling through Nfκβ and AP-1 transcription factors. ³⁸ In the colon, granulocytes and monocytes likely represent the main producers of IL-23, which can lead to the induction of IL-1, IL-17, IL-6, and TNF-α, all strong proinflammatory cytokines. ^{38 39} Through its downstream mediators, IL-23 plays a role in the induction of Th7 cells, which are indicated in bacterial infections, autoimmunity, and IBD. ³⁹

In Cowardin et al showed that as a pair, toxins A and B did not increase IL-1β and IL-23 from bone marrow-derived dendritic cells (BMDCs), but when in combination with bacterial filtrates, PAMPS, or danger associated molecular patterns (DAMPs), they were able to significantly increase the levels of these proinflammatory cytokines. This is likely due to the necessary inflammasome priming signal through TLRs or other PRRs before activation of the inflammasome by the toxins. This was true, as well, for non- C. difficile associated PAMPs as well, such as LPS, which signals through TLR4. In addition to the increase in release of these cytokines, the toxins alone, without the presence of additional PAMPs or DAMPs, were able to increase the transcription of il23 . This suggests that the increase in production of IL-23 may not only be because of an increase in inflammasome signaling following both the priming and activating steps, but also due to direct actions through transcription factors, such as Nfkβ that IL-23 can activate through MAPkinases.

It was observed that IL-1β signaling through IL-1R was responsible for the induction of IL-23, as it was seen that IL-23 levels were significantly reduced when either IL-1R or IL-1β itself were neutralized or nonfunctional. Additionally, supplementing recombinant IL-1β to BMDCs was sufficient to induce IL-23 production when given with bacterial filtrates lacking toxins A or B (which are unable to stimulate IL-23 release). This suggests that toxins A and B stimulate an increase in inflammasome activation, leading to the robust production of IL-1β, which acts downstream, through its receptor IL-1R to induce IL-23 release. This work was correlated in humans by observing serum levels of IL-1β in diarrheal patients with and without CDI compared with nondiarrheal controls. While IL-23 levels were undetectable in human patients, those with diarrhea, regardless of causative agent, had increased IL-1β, suggesting strong inflammasome signaling upstream of IL-23 release, could be pathogenic in diarrheal patients.

CDT and Increased Virulence

The ADP-ribosylating, binary toxin, CDT, has become more prevalent in recent decades and is correlated with increased mortality and morbidity during C. difficile infection. ⁹ Found in upwards of 20% of clinical isolates, CDT-producing C. difficile in research settings is often modeled by two PCR (polymerase chain reaction)-Ribotype 027 strains, either M7404 or the epidemic strain R20291. While both express toxins A and B, as well as CDT, R20291 originated from an outbreak in the United Kingdom, and M7404 from Canada. ^{40 41 42} CDT contains two active subunits, CDTa and CDTb those perform different roles during cellular intoxication. ⁴³ CTDb forms a heptamer and binds with the lipolysis-stimulated lipoprotein receptor (LSR) on the surface of colonic cells, this then allows for the CDTa subunit to bind to the CDTb heptamer and be endocytosed into the cell. ^{44 45} Following this, endosomal acidification activates the CDTb heptamer to insert itself into the endosomal membrane and allows for CDTa to be released into the cytoplasm CDTa is then able to disrupt actin filament elongation, leading to cytoskeleton collapse and formation of structures known as microtubule protrusions, which are thought to increase the adherence of C. difficile to host cells. ⁴⁴

Through in vivo mouse studies comparing mortality and morbidity between R20291 CDT ⁺ strains and isogenic mutants lacking either CDTa or CDTb, it was observed that the presence of CDT significantly increased the severity and disease-associated mortality. Along with this, the presence of CDT increased proinflammatory cytokines, such as IL-1β and IL-6, as well as increased NFκβ activation. Although IL-8 and IL-23, two other proinflammatory cytokines that are positively associated with increased disease severity, were not characterized, increased presence of IL-1β indicating activation of the inflammasome which is a hallmark of infection with toxin A and B producing strains. Additionally, through intoxicating BMDCs, it was observed that CDT, when administered with TcdA/B, as a pair, led to a significant increase in IL-1β production. When considered with previous data showing that TcdA/B were able to activate the inflammasome after it had been primed from upstream PAMPs or DAMPs, it suggested the CDT is an efficient inflammasome primer. Thus, one of the mechanisms through which CDT increases virulence of C. difficile strains is by increasing the activation of the inflammasome, leading to abundant downstream pathogenic cytokines and inflammation.

Activation of NFκβ and priming of the inflammasome require the recognition of pathogenic molecules, often PAMPs including LPS and flagellin, or DAMPs, which include cytosolic DNA and RNA, or extracellular ATP. ^{38 46} Recognition of these molecules often occurs through PRRs, such as TLRs. ¹⁷ Through in vivo and in vitro experiments with TLR2, 4, and 5 knockouts and incubation with CDT and toxins A and B, Cowardin et al observed that the absence of TLR2 abrogated the ability of cells to respond to CDT through Nfκβ signaling and IL-1β production. Additionally, loss of TLR2 in vivo abrogated the increased mortality due to CDT. This suggested that CDT was able to prime the inflammasome through recognition by TLR2, and that this was contributing to the increase in pathogenic inflammation caused by abundant inflammasome activation.

Beyond promoting increased inflammasome activation and inflammatory cytokine release, CDT may additionally increase disease severity by causing apoptosis of eosinophils in a TLR2-dependent mechanism. To be discussed in the next section, eosinophils provide protection during CDI, and their loss due to CDT presence may contribute to the increased host morbidity and mortality of these highly virulent strains.

Eosinophils and CDI

Historically, neutrophils are considered the most prominent responding cell type during CDI. ⁴⁷ They contribute to strong antimicrobial responses, but can also induce bystander tissue damage. ¹⁸

Recently, however, a lesser known innate immune cell population, eosinophils, have been implicated as a core factor in the response to C. difficile infection both in models of CDT ⁺ R20291 strains and non-CDT producing VPI10643 strains, as well as in clinical patients. ^{9 11 12} Like neutrophils, eosinophils are multinucleated granulocytes, but whose roles are most commonly associated with helminth infection, allergies, and asthma. ^{15 22 48} Major functions of eosinophils include the degranulation of molecules, such as major basic protein (MBP), eosinophil peroxidase (EPO), oxygen radicals, and release of molecules, those can degrade the extracellular matrix. ⁴⁹ These functions target foreign antigens and lead to tissue inflammation and remodeling that aims to kill infiltrating pathogens and repair the large amount of tissue damage they often cause. ⁵⁰

In the case of C. difficile infection, patient eosinophilia correlates strongly with better survival and decreased morbidity during disease. While the mechanism through which eosinophils provide protection is not entirely known, their increase due to administration of recombinant IL-25 in mice correlated with decreased clinical scores, suggesting they may play a role in mediating the severity of disease. ¹¹ Additionally, IL-25 supplementation correlated with an increase in IL-4 levels, mucin production in the epithelium of the GI tract, and decreased tissue damage. However, abrogating IL-4 signaling, as well as observing no change mucin production in mice lacking eosinophil, in combination with IL-25 administration, suggested that these factors were not the mechanism through which eosinophils provided protection. Alternatively, mice models lacking eosinophils, regardless of IL-25 administration, had increased epithelial damage as observe by histology, and increased luminal albumin and colon shortening, indicators of severe colitis and tissue damage. Through these results, it is implicated that eosinophils protect through some mechanism that decreases intestinal epithelial damage. Not only observed in mouse models, data collected from CDI+ patients observed that those with undetectable eosinophil counts at time of admission were at increased risk for in-hospital mortality, sepsis, and emergency total colectomy. ¹²

As introduced in the previous section, CDT was observed to cause increased eosinophil apoptosis during CDI, as observed by increased annexin-V staining. ⁹ This reduction in eosinophils was correlated with increased weight loss and mortality in mice during CDI. In mice models lacking eosinophils, infection with CDT ⁺ R20291 strains caused no increased virulence over infection in WT mice, but infection with CDTb ⁻ R20291 (which normally significantly reduces mortality) in mouse models lacking eosinophils increased mortality to levels found in CDT ⁺ strains, suggesting that eosinophil loss contributes to the high virulence found in CDT ⁺ strains. The mechanism through how CDT causes increased eosinophil apoptosis was determined to be through TLR2 signaling; as discussed in the previous section, CDT was seen to increase IL-1β production in BMDCs when in combination with toxins A and B, suggesting that it was a strong primer for the inflammasome. ⁴ In conjunction to the observation that mice lacking TLR2 were significantly protected from CDI-mediated mortality, these mice also had increased levels of eosinophils, which prompted the hypothesis that TLR2 signaling may be playing a role in eosinophil apoptosis due to CDT. Interestingly enough, mice given transfers of eosinophils lacking TLR2 were significantly protected from C. difficile infection, even in the presence of CDT. However, CDT was unable to kill eosinophils in vitro even when TLR2 signaling was intact. These results indicate that while TLR2 signaling seems to be pathogenic during CDI and contributes to eosinophil apoptosis due to CDT ⁺ producing strains, the exact mechanism through how CDT-TLR2 signaling causes eosinophil cell death is not clear, and may be due to indirect methods that relies on the altered inflammatory environment.

Conclusion

Infection with C. difficile (CDI) leads to a robust inflammatory response and results in large amounts of colon epithelial damage, fluid loss, and host morbidity and mortality. Antibiotic usage is the strongest correlated risk factor for CDI and may predispose patients through mechanisms that involve both a reduction in colonization resistance and modulation of a protective immune response, as implicated in the reduction in the cytokine IL-25. The main toxins of C. difficile , toxins A and B, lead to strong inflammasome signaling resulting in IL-Iβ-dependent IL-23 release. The presence of IL-23 can be pathogenic during CDI, especially in high concentration, as it results in the production of other proinflammatory cytokines and the recruitment of inflammatory cells. Additionally, the presence of the binary toxin, CDT, has been shown to increase inflammasome activation through TLR2-mediated priming. This activation of TLR2 by CDT is pathogenic in C. difficile infection and results in proinflammatory cytokine release and increased eosinophil apoptosis. The loss of eosinophils due to CDT is problematic, as eosinophils are protective during infection through mechanisms that likely involve decreasing epithelial damage. Through eosinophils, IL-25 has shown to be protective during disease, as its supplementation results in an increased recruitment of these innate immune cells.

Overall, the immune response to C. difficile is complicated and involves crosstalk between different immune pathways. Through inflammasome signaling, the release of proinflammatory cytokines such as IL-1β and IL-23, and neutrophil recruitment, the immune response likely works to decrease bacterial load and clear the pathogen rapidly. Conversely, through type-2 signatures, such as IL-25, IL-4, and eosinophils, the immune response may be mediating the strong inflammatory profile, and attempting to limit and repair the high levels of tissue damage that are associated with CDI. Together, this shows a coordinated immune response that relies on a careful balance to both promote pathogen clearance and limit host damage.