The innate defense mechanisms against C. difficile infection include the endogenous microbial flora, the mucus barrier, intestinal epithelial cells, and the mucosal immune cells.

C. difficile primarily produces toxin A and toxin B (with certain strains also producing binary toxins), causing disruption to the intestinal epithelium, leading to activation of the immune responses in the lamina propria of the colon. These toxins have a profound impact on innate immune defenses, triggering the release of various proinflammatory mediators, such as cytokines and chemokines, promoting the recruitment and activation of diverse innate immune cells (Figure 1). Further, the disrupted mucosal barrier, due to toxin-mediated damage, allows commensal bacteria to translocate into the lamina propria and systemic circulation, leading to robust inflammatory responses.

In response to C. difficile toxins and their associated damage, intestinal epithelial cells, and innate immune cells in the lamina propria release proinflammatory cytokines (IFNγ, IL-12, IL-6, IL-23, IL-1β, etc.) and chemokines (CXCL1, CXCL2, and CXCL5), leading to the recruitment of neutrophils to the site of infection [35,36,37,38].

Proteins involved in the signaling of the innate immune response, such as nucleotide-binding and oligomerization domain 1 (NOD1), myeloid differentiation factor 88 (MyD88), and adaptor protein for inflammasome, known as apoptosis-associated speck-like protein containing a CARD (ASC), play a role in C. difficile pathogenesis. Studies show that mice deficient in Nod1, MyD88, and ASC signaling have decreased levels of CXCL1 and infiltrating neutrophils in their colons, which is associated with enhanced disease severity and mortality, compared to wild-type mice [33,39,40]. It is worth mentioning that the role of neutrophils and their effect on host factors in CDI susceptibility is complex and context-dependent, with various pathways and cell types involved in the overall pathogenesis. For example, Jarchum et al. found that depletion of neutrophils in mice through antibody-mediated Gr1⁺ (Ly6G) depletion increases the disease severity and mortality during CDI [33]. This finding is supported by the clinical observation that in hospitalized leukemia and allogeneic hematopoietic stem cell transplant patients, neutropenia is positively correlated with CDI susceptibility and recurrent CDI [41,42]. In contrast, another study found that blocking of neutrophil infiltration in the colon by anti-CD18 (leukocyte adhesion molecule) treatment in rabbit leads to decreased Toxin A-mediated enterotoxicity, compared to non-anti-CD18 treated mice [43]. Similarly, another study in a mouse model of CDI showed that mice treated with macrophage migration inhibitory factor (MIF) blocking antibody demonstrated improved disease severity and survival, which is associated with reduced neutrophil recruitment in the colon [44]. However, a recent study by Chen et al. found no differences in CDI severity when they depleted neutrophils using anti-Ly6G antibodies [34]. The differing roles of neutrophils in CDI outcomes in various studies may result from variances in animal models, C. difficile strains, experimental design, and host-specific factors. Other innate immune cells, such as eosinophils, play a protective role in CDI. In studies using mice, Buonomo et al. show that mice adoptive transferred of eosinophils are more protected against CDI [31], supported by a clinical study showing that, in patients, the eosinopenia in peripheral blood at the time of CDI diagnosis was associated with higher mortality [45].

C. difficile and its toxins can activate surface and intracellular innate immune sensors, including TLR4, TLR5, and inflammasome signaling pathways [39,46,47,48]. Depletion of TLR4-signaling pathways in mice results in an increased bacterial burden and disease severity during CDI [46]. On the other hand, TLR5 deficiency did not affect survival, but stimulating TLR5 with flagellin provided protection against CDI, likely due to its positive impact on the intestinal epithelial layer [47].

In vitro, infection of murine peritoneal macrophages with a toxigenic strain of C. difficile results in the release of the proinflammatory cytokine pro-IL-1β, a process that is dependent on MyD88 and, to some extent, TLR2 [49]. In the same study, toxigenic C. difficile activated inflammasome through the ATP-P2X₇ pathway, leading to caspase-1-dependent pyroptosis [49]. In a different study, stimulating the J774A.1 murine macrophage cell line with C. difficile’s surface layer protein (SLP) led to increased expression of TLR2, TLR4, and MHCII [50]. Furthermore, the activation of TLR4 using SLPs triggered p38 signaling, leading to increased production of IL-1β, IL-6, TNF-α, IL-12p40, and the upregulation of chemokines like MIP-1α, MIP-2, and MCP [50].

Recent studies have shed light on the protective role of innate lymphoid cells (ILCs) in acute CDI. Unlike adaptive immune cells like T and B cells, ILCs, which include ILC1, ILC2, and ILC3, lack antigen-specific receptors [51]. However, they possess the ability to detect cytokines and chemokines and are pivotal in orchestrating immune responses against infections and maintaining tissue homeostasis [51]. During CDI, when commensal microorganisms, pathobiont bacteria, or C. difficile toxins translocate across the gut barrier into deeper tissues, this triggers the activation of ILCs. A study showed that Nfil3-deficient (Nfil3^-/-) mice, which exhibit defects in the development and functionality of ILCs, demonstrated heightened susceptibility to acute CDI compared to their wild-type counterparts [52]. Nfil3^-/- mice infected with C. difficile manifest increased weight loss and higher mortality rates than their wild-type (WT) counterparts. Another study conducted by Abt and colleagues uncovered the protective function of IFNγ-expressing ILC1 as part of the host’s defense against CDI, complemented by the role of IL22-expressing ILC3 [53]. Additionally, another research group has demonstrated the role of ILC2s in protection against C. difficile colitis. For instance, Frisbee et al. showed that ILC2 provides defense against acute CDI, especially when activated by IL-33 [32]. Notably, treating C. difficile-infected mice with IL-33 leads to reduced neutrophil levels and increased eosinophils in the colon, resulting in type 2-associated mucosal immunity. Similarly, another study further underscored the IL-25-mediated role of ILC2s in defending against CDI [31]. In parallel, other researchers also demonstrated the protective role of ILC3 in safeguarding against CDI, primarily through the interleukin-22-mediated effect [54,55].

The aforementioned studies indicated the crucial role of innate immunity in the initial containment of infections and its contribution to resolving CDI. Nonetheless, the recurrence of episodes implies that adaptive immune cells may be fundamentally involved. This is attributed to their capacity to respond in an antigen-specific manner and generate memory, thereby providing additional protection.

The defining feature of the adaptive immune system is the clonal expansion of lymphocytes, resulting in a durable and highly specific response. Typically, memory T and B cells orchestrate more robust and rapid immune reactions to pathogens—rapidly multiplying, generating effector cytokines, and executing various effector functions [56]. However, an impaired immune response to pathogens can result in recurring infections.

Around 20 to 35% of individuals treated for CDI encounter at least one additional episode within 2 to 8 weeks of their initial CDI treatment [18,42]. These subsequent events can manifest as either a relapse with the same C. difficile strain or as a reinfection with a different strain [57]. One possible factor contributing to susceptibility to recurrent CDI is the ongoing disturbance of gut microbiota diversity [15,58]. This, in conjunction with a weakened host response, may play a role, since low levels of serum antibodies against toxins A and B have been linked to the CDI recurrences [59,60].

In CDI, immunoglobulins such as systemic IgG and mucosal IgA significantly influence disease outcomes. In human CDI cases, the disease’s severity shows an inverse correlation with the levels of toxin-specific IgA and IgG antibodies in the serum and secretory intestinal IgA [60,61]. Furthermore, in fecal samples, individuals experiencing a single CDI episode demonstrated significantly higher anti-TcdA IgA titers, compared to those with recurrent CDI [62]. Studies also have found an inverse relationship between IgG levels against toxin B and disease severity, as well as CDI recurrence. Notably, patients possessing antibodies against toxin B exhibit enhanced protection compared to those with antibodies against toxin A [63]. Further, monoclonal antibodies targeting TcdB have demonstrated greater efficacy in CDI treatment [21], indicating TcdB’s higher toxicity compared to TcdA. It is important to note that while elevated antibodies against C. difficile toxins can guard against the disease and its relapse, they do not prevent C. difficile colonization in the colon.

Most of the research pertaining to the host’s adaptive immunity in CDI has primarily concentrated on humoral responses. On the other hand, the importance of T follicular helper (Tfh) cells, which play a critical role in generating plasma cells that produce antibodies and long-lasting memory B cells, has only recently begun to be investigated. The exploration of various subsets of T cells and their effector functions against C. difficile is still in its early stages.

At the germinal center, Tfh cells play a crucial role in aiding B cells, ultimately leading to the differentiation of activated B cells into plasma cells and memory B cells [64,65]. Consequently, Tfh cells are instrumental in conferring antibody-mediated protection to the intestinal mucosa against pathogens. In a murine model of recurrent C. difficile infection, Amani et al. observed a notable expansion in the population of lymph node resident Tfh cells, encompassing both germinal center and non-germinal center Tfh cells, following CDI when compared to uninfected mice [66]. Despite the observed expansion of Tfh cells, the B cell response proved insufficient in preventing disease recurrence. Furthermore, the authors demonstrated that CDI failed to elicit a robust B cell memory response.

Studies have demonstrated that C. difficile strains trigger a CD4⁺ T cell response. For example, the hypervirulent C. difficile R20291 strain elicits a robust Th1 and Th17 response, as evidenced by an increased presence of IFNγ⁺ and IL-17A⁺ CD4⁺ T cells, when compared to the non-virulent C. difficile 630 strain in co-culture with murine splenocyte and bone-marrow-derived dendritic cells [67]. In a clinical study, C. difficile-infected patients exhibited a shift in immune responses from Th1 to Th17 or Th2 as disease severity increased [68]. Studies by other investigators also demonstrated the role of T cells in CDI. Notably, young children display resistance to CDI, and a study in mice has highlighted the role of IL-17A produced by γδ T cells in this resistance [34]. Neonatal mice, resistant to CDI, demonstrated substantial IL-17 production by RORγt⁺ γδ T cells [34]. However, this protective effect was lost upon depletion of these IL-17-producing T cells.

Regulatory T cells (T_reg), a subset of CD4⁺ T cells, are crucial for maintaining intestinal immune tolerance and homeostasis. Their specific impact on a host’s susceptibility to acute CDI and relapse remains unclear. However, a recent study highlighted the vital role of T_reg cells in successfully engrafting fecal microbiota and clearing chronic CDI in mice [69]. Mechanistically, depleting T_reg cells causes an exaggerated immune response in the colon, hindering C. difficile clearance by impeding the engraftment of microbial populations derived from fecal microbiota transplantation (FMT).