Clostridioides difficile infection (CDI) is currently the leading cause of nosocomial diarrhea associated with the use of antibiotics (Brazier, 1998; Zhang et al., 2016). C. difficile is a Gram positive opportunistic pathogen that takes advantage of gut dysbiosis to establish colonization of the intestine, inducing tissue damage by toxin production. C. difficile is responsible for about 250,000 CDI cases and 13,000 deaths in the United States each year (Center for Disease Control and Prevention, 2019). Given the increase in the incidence and severity of CDI, the high rate of recurrence (>35%) and the appearance of hypervirulent strains, the Center for Control and Prevention in the United States has pointed this infection as an emergency (McDonald et al., 2007; CDC, 2012; Burke and Lamont, 2014; Balassiano et al., 2016; Elliott et al., 2017). Furthermore, there has been a large increase in community-acquired cases in the last decades, affecting individuals without risk factors typically associated with CDI (Legaria et al., 2003; Chitnis et al., 2013; Gupta and Khanna, 2014). Of importance, since the onset of the pandemic, approximately 72% of COVID-19 patients have been treated with broad-spectrum antibiotics. Therefore, the number of C. difficile infections, particularly recurrences, is expected to increase over time (EBR, 2020). All this together exhibit the urgent nature of this pathogen.

The immune response against C. difficile has been assessed before, however many aspects require a deeper understanding (Hernández Del Pino et al., 2020). Particularly, intestinal macrophages demand constant renewal from circulating monocytes, shaping the composition of the macrophage compartment in the intestinal mucosa when infection, inflammation or trauma alter tissue homeostasis (Bain and Mowat, 2014b; Na et al., 2019). Intestinal macrophage populations are heterogeneous and display different functions. Both anti-inflammatory and pro-inflammatory macrophages of the gut wall derive from blood monocytes that enter the mucosa and mature locally (Bain and Mowat, 2014a; Bain and Mowat, 2014b). In experimental CDI models, macrophages produce pro-inflammatory cytokines and chemokines, as well as show expression of certain receptors and molecules that indicate macrophages activation (Vohra and Poxton, 2012; Collins et al., 2014). Besides, the interaction and internalization of C. difficile spores and toxins has been observed in monocytes, macrophages or epithelial cells (Solomon et al., 2005; Modi et al., 2011; Paredes-Sabja et al., 2012; Chen et al., 2020). To our knowledge, the internalization of vegetative bacteria has not been demonstrated yet.

Platelets have aroused great interest in recent years, proving to be key participants in numerous immunological processes. Beyond their central roles in thrombosis and hemostasis, platelets play multiple roles in host defense against infection, interacting with bacteria, protozoa, fungi and viruses and showing anti-microbial properties (Klinger, 1997; Yeaman, 1997; Lam et al., 2015). Platelets can also interact with innate and adaptive immune cells promoting neutrophils activation, cytokines secretion by monocytes, leukocytes migration to the site of inflammation, B cells differentiation and immunoglobulin classes switching (Klinger, 1997; Yeaman, 1997; Trzeciak-Ryczek et al., 2013; Maouia et al., 2020; Scherlinger et al., 2023). In the context of CDI, severe infection could lead to sepsis and extra-intestinal infection (Lowenkron et al., 1996; Libby and Bearman, 2009; Steele et al., 2012; Abid and Bischof, 2019; Olsen et al., 2023). Moreover, inflammation in the gut can induce platelet activation and migration (Gaertner et al., 2017; Shannon, 2021; Trivigno et al., 2023), then platelets attached to the activated endothelium promote monocyte migration (Huilcaman et al., 2022; Trivigno et al., 2023). Beneficial or detrimental roles related to clinical symptoms have been reported for platelets counts during CDI (Pant et al., 2009; Byrn et al., 2012; Lee et al., 2014; Yan et al., 2017; Mihăilă et al., 2019; Nseir et al., 2019; Allegretti et al., 2020; Phanchana et al., 2020; Zhao et al., 2020; Buchrits et al., 2021). Nevertheless, their modulatory functions on immune responses against C. difficile have not been addressed.

Our findings provide new insights into the role of macrophages and platelets during CDI, which could be useful for the development of new therapeutic approaches in search of replacing antibiotics or as treatment adjuvants.

Clostridioides difficile infection (CDI) is prevalent in hospitalized patients, and despite prevention activities, CDI remains a life-threatening disease, highlighting the need for new treatment approaches and a better understanding of host-pathogen crosstalk.

It is now well-known that innate immunity is a double-edged sword during CDI, since it is crucial for inducing intestinal inflammation but also for resolving infection (Hernández Del Pino et al., 2020; Nibbering et al., 2021). Regarding macrophages, most of the reports are based on cell lines or murine models and evidence the activation of macrophages in response to C. difficile. Macrophages produce proinflammatory MIP-1α, MIP-2, MCP, IL-1β, IL-6, TNF-α, IL-12p40, and IL-12p70 when stimulated with C. difficile antigens (e.g. surface layer proteins, flagella, HSP42, and HSP60) or live bacteria (Vohra and Poxton, 2012; Collins et al., 2014; Saad et al., 2023). Likewise, these phagocytes up-regulate TLR2, TLR4, CD40, CD80, and MHCII expression (Collins et al., 2014). Moreover, inflammasome activation via TLR2-ATP-P2X7-Caspase 1 has been proposed as a beneficial mechanism to control bacterial load (Liu et al., 2018).

In our work we employed human monocyte-derived macrophages to address the endocytic capacity of these cells. Despite the fact that the intracellular localization of C. difficile has not been directly demonstrated yet, it has been proposed that phagocytosis of the bacteria or its toxins is essential to activate macrophages (Liu et al., 2018). C. difficile spores can be internalized by murine macrophages but they manage to survive and avoid their clearance (Paredes-Sabja et al., 2012). C. difficile toxin A can remodel cell membranes and gain access to interact with TLR9 in epithelial cells (Chen et al., 2020). Taking into account this information and the importance of the vegetative bacteria to induce pathogenesis at local level, we performed our experiments with the vegetative form of the C. difficile hypervirulent epidemic strain BI/NAPI/027 and the non-toxigenic CD160 strain. We found that human macrophages internalized C. difficile both at 1 or 24h of in vitro culture, showing greater uptake levels when incubated with higher amounts of bacteria. Our work is one of the first using different live C. difficile strains in normoxic in vitro models. We stained both live and inactivated C. difficile with FITC using a short time and few-steps protocol, guaranteeing bacterial viability. We believe that this could constitute a powerful tool for future research about the role of immune and non-immune cells in disease conditions.

The partnership between macrophages and platelets has been addressed in arteries pathologies (Mansilla et al., 2008; Mehrpouri et al., 2019; Huilcaman et al., 2022) and infections (Honda et al., 2009; Feng et al., 2014; Wong et al., 2016; Ali et al., 2017; Carestia et al., 2019; Rossaint et al., 2021). In the context of CDI, the studies are scarce and generally evaluate the importance of the platelet count in the severity of the disease. Some reports propose that an increased platelets number is associated with a better prognosis (Byrn et al., 2012; Lee et al., 2014; Yan et al., 2017; Allegretti et al., 2020; Zhao et al., 2020), while other studies associate this to worse outcomes (Albrich and Rimland, 2007; Pant et al., 2009; Mihăilă et al., 2019; Nseir et al., 2019; Phanchana et al., 2020; Buchrits et al., 2021). In severe cases of C. difficile infection, the intestine fails in its function as a barrier to both intestinal bacterial and toxins, allowing them to reach the portal circulation and developing extra-intestinal CDI (Lowenkron et al., 1996; Libby and Bearman, 2009; Steele et al., 2012; Abid and Bischof, 2019; Olsen et al., 2023). Moreover, vascular changes ligated to C. difficile-induced inflammation have been demonstrated as well as increased colonic vascular permeability and angiogenesis (Kurose et al., 1994; Huang et al., 2019). Platelets attached to the activated endothelium promote the migration of monocytes and can also modulate the composition of the intimal layer of blood vessels which favors monocytes migration (Huilcaman et al., 2022; Trivigno et al., 2023). With the end to get a better understanding of platelets role on human macrophages responses during CDI, we assessed the intercommunication of platelets with C. difficile and innate immune cells.

Platelets can directly sense microorganisms (Houlihan and Copley, 1946). Platelets can migrate to sites of infection and aggregate with the infiltrating bacteria, in addition to collaborating with the immune system (Gaertner et al., 2017; Shannon, 2021; Trivigno et al., 2023). The ability of platelets to recognize, collect and/or bound pathogens has been described for E. coli (Gaertner et al., 2017; Palankar et al., 2018), S. aureus (Gaertner et al., 2017), L. monocytogenes (Gaertner et al., 2017), B. abortus (Trotta et al., 2018), and even retroviruses (Zucker-Franklin et al., 1990). Then, bacteria sensing by platelets is not restricted to a particular type of microorganism, since both live and inactivated, Gram positive and Gram negative, opsonized or not bacteria can be recognized. In line with these works, we evidenced a direct interaction of platelets with C. difficile. Platelets also act as covercytes, spreading on the foreign microorganism to cover and seal it (White, 2005). No specific molecule for the interaction between platelets and bacteria has been identified. Rather, it has been proposed that physical forces that help the platelets become “sticky” are responsible for platelet-bacteria interaction. Accordingly, we observed changes in platelets shape which could be related to actomyosin system that shapes the spreading and contraction dynamics of platelets. Besides, we described the establishment of monocytes-platelets complexes in fresh blood, which were induced in the presence of the bacteria. In accordance, monocytes-platelets crosstalk has also been observed for tetanus toxoid (Gudbrandsdottir et al., 2013) and B. abortus (Trotta et al., 2018), in sepsis (Gawaz et al., 1995), in Dengue patients (Hottz et al., 2014) and in non-infectious contexts (Weyrich et al., 1996; Weyrich et al., 2005). We also showed that platelets formed complexes with macrophages when co-cultured together both with or without C. difficile (always over 70%). Interestingly, platelets were not only attached to macrophages surface but they also localized intracellularly. In contrast to our findings, Trotta and col. observed that platelets surrounded specifically B. abortus infected monocytes/macrophages (Trotta et al., 2018). On the other hand and in line with us, Carestia and col. identified platelets/macrophages associations both in LPS presence or absence (Carestia et al., 2019). Altogether, the previous reports and our results reveal platelets versatility, acting both as pathogens patrollers and as links to the immune system.

Platelets and platelets-secreted PAF induce the endocytosis of B. abortus (Lee et al., 2016; Trotta et al., 2018) and S. aureus (Ali et al., 2017) by monocytes and macrophages. In line with this, we demonstrated that human platelets promote C. difficile internalization. It has been proposed that platelets act as carriers of B. abortus, delivering bacteria to monocytes/macrophages (Trotta et al., 2018). Unlike this report, we found an increment in macrophages uptake capacity when platelets were pre-incubated with C. difficile and then added to macrophages culture as well as when platelets were cultured with C. difficile and macrophages together. These results suggest that platelets modulate macrophages ability rather than being transporters of C. difficile. Our experiments with live NAP1/BI/027, live CD160 or CDH plus the NAP1/BI/027 secretome showed no differences when comparing with CDH stimulation, suggesting that the bacteria are not releasing factors that modify the interactions between macrophages and platelets. The obtained results might us speculate that platelets-macrophages physical contact is required to enhance C. difficile internalization. Nevertheless, we cannot rule out the possibility that platelets are releasing soluble factors that activate macrophages in a very early way. Although the underlying mechanisms may differ, the outcome is the same: the enhancement of the uptake potential.

To date, it seems that platelets participation against bacteria contribute to develop a protective response as well as to spread infection. Platelets were proposed as rapid defenders against infections mediated by blood-borne pathogens such as B. cereus and S. aureus (Wong et al., 2016; Wuescher et al., 2016) and a beneficial role for the host has also been suggested in Brucellosis (Trotta et al., 2018; Trotta et al., 2020). On the contrary, S. pyogenes dissemination has been related to the presence of platelets (Kahn et al., 2013). Our confocal micrographs show what appears to be processed C. difficile in the presence of platelets, suggesting that the interaction of platelets with macrophages could lead to bacteria clearance. Nevertheless, whether the bacteria are destroyed and whether platelets could promote C. difficile elimination deserve further investigation.

Different mechanisms can be explored to internalize pathogens. Macropinocytosis, a clathrin-independent endocytic process that internalizes extracellular fluid, nutrients and antigens, has been involved in the uptake of several infectious agents by a broad variety of cell types (Young et al., 1989; Watarai et al., 2001; Niebuhr et al., 2002; Watarai et al., 2002; Mercer and Helenius, 2009; García-pérez et al., 2012; de Carvalho et al., 2015; Nam et al., 2017; Chang et al., 2021). C. difficile seems not to be an exception since here we showed that only amiloride (an ion exchange inhibitor) reduced the percentage of C. difficile-internalizing macrophages. Amiloride is a selective Na+/H+ antiport inhibitor but how it inhibits macropinocytosis is not yet fully understood. Some proteins of Rho family GTPases (e.g. Rac1 and Cdc42) have been implicated during macropinocytosis (Koivusalo et al., 2010). PLC-PKC-Nox2 pathway has also been described to regulate macropinosome formation, membrane ruffling and fluid phase uptake (Espinal et al., 2022) while Glycogen synthase kinase 3 (GSK3) was proposed as negative regulator by altering Wnt-β-catenin signaling (Tejeda-muñoz et al., 2019; Albrecht et al., 2020). When amiloride was added in the presence of platelets, the reduction in the internalization capacity did not reach basal levels, suggesting that other mechanisms different to macropinocytosis could have a role in C. difficile uptake. Likewise, this could also indicate that the presence of platelets per se could decrease the effect of amiloride exerted on macrophages. Importantly, macrophages can play constitutive macropinocytosis, which could serve as an ideal means for delivering C. difficile antigens to intracellular PRRs. This is especially relevant at sites of continuous contact with non-self material such as the gut (Canton, 2018), highlighting the relevance of macropinocytosis during CDI.

To the best of our knowledge, this is the first study elucidating the role of human macrophages and platelets in the immune response against C. difficile. Although our assays were performed in vitro, they provide the basis for proposing platelets as sentinels and modulators of the immune response during CDI. Conventionally, endocytic processes are efficient ways to eliminate invading pathogens, but pathogens can also develop strategies to perpetuate the infection. So many questions still required to be answered. Is C. difficile destroyed within macrophages? Are platelets promoters of macrophages microbicidal functions? or do they induce a C. difficile reservoir? Is C. difficile processed and presented to activate T cells? Can platelets directly eliminate C. difficile? To unravel these, further studies are required.

In conclusion, we evidenced that vegetative form of C. difficile is internalized by human macrophages. Platelets could directly interact with monocytes and macrophages as well as with bacteria. Moreover, C. difficile induced the establishment of monocytes-platelets complexes. Platelets also modulated C. difficile uptake mediated by macrophages, enhancing the internalization capacity of the phagocytes. We finally determined that the uptake of C. difficile was mainly mediated by macropinocytosis.

These results highlight the role of innate immune cells in CDI, describing new cellular mechanisms involved in the internalization of C. difficile. Furthermore, we put in discussion the importance of macropinocytosis in pathogens sensing, a role that is ordinarily underappreciated in the surveillance mechanisms of innate immune cells.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The studies involving humans were approved by Ethics Committee of UNNOBA (COENOBA). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

AB and VP conceived and designed the experiments. AB performed the experiments with RHDP contribution. AB, and FF analyzed the data. PB and RHDP helped with the discussion and the experimental design. AB and VP wrote the first draft of the manuscript. All authors contributed to manuscript reading and revising and approved the submitted version.