, 2009), it is not limited to the hospital environment; community-acquired CDI and asymptomatic carriage are also prevalent (Limbago et al., 2009; Freeman et al., 2010). Production of toxins and spores is the most important virulence determinant of C. difficile. The toxins are highly immunogenic. They have been shown to induce the production of pro-inflammatory cytokines such as IL-1α, IL-1β, IL-6, IL-8 and TNF-α (Flegel et al., 1991; Linevsky et al., 1997; Melo Filho et al., 1997; Johal et al., 2004; Canny et al., 2006) and are responsible for the acute inflammatory response in C. difficile infection (Savidge et al., 2003), which is characterized by pseudomembrane click here formation (Knoop et al., 1993; Castagliuolo &

LaMont, 1999). However, a large number of proteins are released along with the toxins during growth of C. difficile (Mukherjee et al., 2002). These include several

surface-associated proteins such as surface-layer proteins (SLPs), flagella, cell wall proteins like Cwp66 and Cwp84, GroEL and fibronectin-binding protein 68, which are involved in adhesion (Hennequin et al., Obeticholic Acid datasheet 2001b; Tasteyre et al., 2001; Waligora et al., 2001; Calabi et al., 2002) and have also been found to elicit immune responses within the host (Péchiné et al., 2005a, b; Wright et al., 2008). Serum IgG to such surface proteins have been detected in patients and healthy adults in several studies (Pantosti et al., 1989; Mulligan et al., 1993; Sánchez-Hurtado et al., 2008), and in many, a correlation between lower levels of antibodies to somatic antigens and the occurrence or recurrences of disease was identified (Mulligan et al., 1993; Kyne et al., 2000; Péchiné et al., 2005a). Interestingly, in one study, the toxins appeared to be less immunogenic than the somatic antigens, suggesting that surface

adhesins were able to induce Digestive enzyme a host immune response during the course of infection and the intensity of this response affected the outcome of infection (Péchiné et al., 2005a). It has thus been suggested that antibodies against toxin as well as nontoxin antigens may determine the outcome of infection with C. difficile (Kelly & Kyne, 2011). These observations have led to the investigation of different surface-associated proteins such as FliD and FliC, SLPs, Cwp84 and Cwp66 as vaccine components (O’Brien et al., 2005; Péchiné et al., 2007, 2011). GroEL and Cwp66 are heat-shock proteins (HSPs) of C. difficile that are strongly induced and expressed on the cell surface following heat shock at 42 and 60 °C, respectively (Hennequin et al., 2001a; Waligora et al., 2001). The primary aim of this study was to assess the production of immunomodulatory cytokines by a macrophage cell line when challenged with different C. difficile proteins. These included SLPs, flagella, HSPs induced at 42 and 60 °C, and cell-free culture supernatants collected at different stages during the growth of C. difficile.