Fasciola hepatica Kunitz Type Molecule Decreases Dendritic Cell Activation and Their Ability to Induce Inflammatory Responses

December Fasciola hepatica Kunitz Type Molecule Decreases Dendritic Cell Activation and Their Ability to Induce Inflammatory Responses Cristian R. Falco n 0 3 Diana Masih 1 3 Gerardo Gatti 1 2 3 Mara Cecilia Sanchez 1 3 Claudia C. Motra n 1 3 Laura Cervi * 1 3 0 Department of Biological Chemistry, Faculty of Chemical Sciences, National University of Cordoba, CIQUIBIC-CONICET , C o rdoba, Argentina, 1 Department of Clinical Biochemistry, Faculty of Chemical Sciences, National University of Cordoba, CIBICI-CONICET , Co rdoba, Argentina, 2 Foundation for the advancement of Medicine , C o rdoba , Argentina 3 Editor: Jagadeesh Bayry, Institut National de la Sante et de la Recherche Me dicale U 872 , France The complete repertoire of proteins with immunomodulatory activity in Fasciola hepatica (Fh) has not yet been fully described. Here, we demonstrated that Fh total extract (TE) reduced LPS-induced DC maturation, and the DC ability to induce allogeneic responses. After TE fractionating, a fraction lower than 10 kDa (F,10 kDa) was able to maintain the TE properties to modulate the DC pro- and anti-inflammatory cytokine production induced by LPS. In addition, TE or F,10 kDa treatment decreased the ability of immature DC to stimulate the allogeneic responses and induced a novo allogeneic CD4+CD25+Foxp3+ T cells. In contrast, treatment of DC with T/L or F,10 kDa plus LPS (F,10/L) induced a regulatory IL27 dependent mechanism that diminished the proliferative and Th1 and Th17 allogeneic responses. Finally, we showed that a Kunitz type molecule (Fh-KTM), present in F,10 kDa, was responsible for suppressing pro-inflammatory cytokine production in LPS-activated DC, by printing tolerogenic features on DC that impaired their ability to induce inflammatory responses. These results suggest a modulatory role for this protein, which may be involved in the immune evasion mechanisms of the parasite. Responses The DC are the main antigen-presenting cells and are essential for initiating an effective adaptive response against diverse pathogens [1]. Unlike bacteria, helminth products do not mature DC in a conventional way, having been shown to possess the ability to suppress TLR-induced DC maturation [2] [3]. F. hepatica is a trematode parasite that causes disease in various ruminants, and is also an emergent disease in humans [4]. Different antigenic preparations from this parasite, such as tegumental or excretory-secretory products (ESP), are all capable of down-modulating TLR-induced DC activation, thus biasing the immune response toward an anti-inflammatory T regulatory/Th2 phenotype [5 7]. Among the excreted-secreted products of this parasite, cathepsin L1 cysteine protease (FhCL1) is one of the predominant secreted proteins, which has been shown to be involved in the down-regulation of LPS-induced macrophage maturation by TLR3 degradation, leading to a reduced TRIF-dependent MyD88independent macrophage activation signaling pathway [8]. However, we have demonstrated that ESP and a somatic extract impair the DC activation induced by TLR ligands that involve both adaptor molecules (TRIF and MyD88) as well as non-TLR signaling [6, 7], suggesting that other molecules distinct from FhCL1 but present in F hepatica antigens modulate DC maturation. Considering that the parasite has to migrate through the host tissues and confront the inflammatory response induced by signals from both the parasite and exogenous environmental, it is reasonable to assume that distinct molecules, secreted or expressed by the parasite in its tegument, may participate during its migration in the prevention of an appropriate activation of DC, resulting in an anti-inflammatory control. In this work, we have demonstrated the capacity of Fh total extract (TE) and a proteic fraction lower than 10 kDa (F,10 kDa) of TE to endow TLR-maturated DC with tolerogenic properties that promote T cell tolerance through an IL-27 dependent mechanism. It was also shown that both TE and F,10 kDa-treated DC were able to induce de novo Foxp3 T reg cells. Finally, we demonstrated that F. hepatica Kunitz serine protease inhibitor, a protein present in TE and in excretory-secretory products, as well as in the tegument of the parasite, is capable of printing regulatory features on TLR-induced activated DC, thereby impairing their ability to induce inflammatory responses. In agreement with our previous studies [6, 7], antigens from F. hepatica negatively modulated the TLR-induced maturation of murine DC from BALB/c by reducing IL-12, IL-6, TNF production (Fig. 1A). Since the maturation status of DC is closely related to the type of adaptive response generated, it is feasible to hypothesize that LPS maturated DC in the presence of TE have less ability to Fig. 1. Proteins lower than 10 kDa from TE modulate mouse and human DC maturation and their allostimulatory capacity. (A) DC from BALB/c mice were incubated with medium, LPS (1 mg/ml), TE (80 mg/ml) or TE plus LPS (T/L) for 18 h and cytokine concentration was evaluated in the supernatants by ELISA. (B) DC from BALB/c mice treated as described in A, were co-cultured for 5 days with C57BL/6 splenocytes at a 1:10 ratio. Allogeneic responses were determined by the proliferative 3[H]-thymidine incorporation assay and IFN-c production by ELISA. (C) hDC from healthy donors treated with medium, LPS (100 ng/ml), TE (80 mg/ml) or T/L for 24 h were cultured with allogeneic PBMC at 1:10 DC:PBMC cell ratio and the proliferative and IFN-c response were tested. (D) IL-12p70, TNF and IL-6 were detected by ELISA in the supernatant of DC treated with medium or F,10 kDa (20 mg/ml) in the presence or absence of LPS for 18 h. (E) Elution profile of proteins lower than 10 kDa from TE, obtained by the ultrafiltration membrane with a cutoff lower than 10 kDa (F,10 kDa). (F) DC from BALB/c mice were treated with medium, F,10 kDa (20 mg/ml) or TE incomplete, without F,10 kDa (TEin, 80 mg/ml), and OVA peptide (0.5 mg/ml) in the presence or absence of LPS (1 mg/ml), before being cultured with splenocytes from DO11.10 TCR-transgenic mice, and IFN-c production was evaluated after 5 days. Data show the mean of at least four wells SD, and are representative at least of two experiments with similar results, with p representing a significant difference in the Students t-test. initiate inflammatory adaptive responses than fully matured DC. Thus, we evaluated the capacity of these cells to initiate allogeneic responses. Although TE treatment did not modify the allogeneic stimulatory ability of immature DC, the T/L-treated DC did indeed reveal less capability than fully matured LPS-DC to induce a proliferative response or IFN-c secretion against allogeneic cells (Fig. 1B), with IL-4 production being undetectable for all treatments (data not shown). In addition, as it had been established that TE treated DC had a reduced ability to initiate inflammatory responses to allo-antigens in culture using murine cells, we now wanted (...truncated)