Fasciola hepaticaFatty Acid Binding Protein Inhibits TLR4 Activation and Suppresses the Inflammatory Cytokines Induced by Lipopolysaccharide In Vitro and In Vivo
Abstract:Toll-like receptor 4 (TLR4), the innate immunity receptor for bacterial endotoxins, plays a pivotal role in the induction of inflammatory responses. There is a need to develop molecules that block either activation through TLR4 or the downstream signaling pathways to inhibit the storm of inflammation typically elicited by bacterial lipopolysaccharide (LPS), which is a major cause of the high mortality associated with bacterial sepsis. We report here that a single intraperitoneal injection of 15μg Fasciola hepa… Show more
“…7C,D). These results indicate that Fh15 down regulates the expression of inflammatory cytokines at similar intensity to that previously demonstrated by native Fh12 12 .Figure 7Fh15 suppressed the expression of TNFα and IL1β cytokines from mouse BMDM and human monocyte cell line (THP1-Blue CD14) in response to LPS. Bone marrow derived macrophages (BMDM) from naive C57BL/6 mice and THP1-Blue CD14 cells were treated with 5 μg/ml or 10 μg/ml Fh15, respectively 30 min before stimulation with LPS (100ng/ml or 1 μg/ml, respectively).…”
Recently, we reported that a native Fasciola hepatica fatty acid binding protein (FABP) termed Fh12 is a powerful anti-inflammatory protein capable of suppressing the LPS-induced expression of inflammatory markers in vivo and in vitro. Because the purification of a protein in native form is, in many situations not cost-beneficial and unsuitable for industrial grade scale-up, this study accomplished the task of optimizing the expression and purification of a recombinant form of FABP (Fh15). Additionally, we ascertained whether this molecule could exhibit a similar suppressive effect on TLR-stimulation and inflammatory cytokine expression from macrophages than those previously demonstrated for the native molecule. Results demonstrated that Fh15 suppresses the expression of IL-1β and TNFα in murine macrophages and THP1 Blue CD14 cells. Additionally, Fh15 suppress the LPS-induced TLR4 stimulation. This effect was not impaired by a thermal denaturing process or blocked by the presence of anti-Fh12 antibodies. Fh15 also suppressed the stimulation of various TLRs in response to whole bacteria extracts, suggesting that Fh15 could have a broad spectrum of action. These results support the possibility of using Fh15 as an excellent alternative for an anti-inflammatory drug in preclinical studies in the near future.
“…7C,D). These results indicate that Fh15 down regulates the expression of inflammatory cytokines at similar intensity to that previously demonstrated by native Fh12 12 .Figure 7Fh15 suppressed the expression of TNFα and IL1β cytokines from mouse BMDM and human monocyte cell line (THP1-Blue CD14) in response to LPS. Bone marrow derived macrophages (BMDM) from naive C57BL/6 mice and THP1-Blue CD14 cells were treated with 5 μg/ml or 10 μg/ml Fh15, respectively 30 min before stimulation with LPS (100ng/ml or 1 μg/ml, respectively).…”
Recently, we reported that a native Fasciola hepatica fatty acid binding protein (FABP) termed Fh12 is a powerful anti-inflammatory protein capable of suppressing the LPS-induced expression of inflammatory markers in vivo and in vitro. Because the purification of a protein in native form is, in many situations not cost-beneficial and unsuitable for industrial grade scale-up, this study accomplished the task of optimizing the expression and purification of a recombinant form of FABP (Fh15). Additionally, we ascertained whether this molecule could exhibit a similar suppressive effect on TLR-stimulation and inflammatory cytokine expression from macrophages than those previously demonstrated for the native molecule. Results demonstrated that Fh15 suppresses the expression of IL-1β and TNFα in murine macrophages and THP1 Blue CD14 cells. Additionally, Fh15 suppress the LPS-induced TLR4 stimulation. This effect was not impaired by a thermal denaturing process or blocked by the presence of anti-Fh12 antibodies. Fh15 also suppressed the stimulation of various TLRs in response to whole bacteria extracts, suggesting that Fh15 could have a broad spectrum of action. These results support the possibility of using Fh15 as an excellent alternative for an anti-inflammatory drug in preclinical studies in the near future.
“…Although helminth infection is associated with an increase in circulating LPS, there are numerous helminth-mediated immunoregulatory mechanisms in place to reduce LPS-associated inflammatory response. For example, helminths infections are beneficial in sepsis by inducing IL-10 expression (6), and Fasciola hepatica secretes fatty acid binding proteins to reduce inflammatory responses (8). Our data suggest hookworm infection also protects from sepsis through hRetn-dependent and hRetn-independent mechanisms.…”
Section: Discussionmentioning
confidence: 77%
“…Recent studies have shown that chronic infection with filarial nematode Litomosoides sigmodontis protects mice from fatal sepsis through TLR2-dependent activation of macrophages and inhibition of proinflammatory cytokines (6). In addition, helminth antigens such as Acanthocheilonema viteae ES-62 and Fasciola hepatica fatty acid binding protein bind to TLR4 and reduce proinflammatory cytokine production (7,8).…”
Helminths trigger multiple immunomodulatory pathways that can protect from sepsis. Human resistin (hRetn) is an immune cell-derived protein that is highly elevated in helminth infection and sepsis. However, the function of hRetn in sepsis, or whether hRetn influences helminth protection against sepsis, is unknown. Employing hRetn-expressing transgenic mice (hTg) and recombinant hRetn, we identify a therapeutic function for hRetn in lipopolysaccharide (LPS)-induced septic shock. hRetn promoted helminth-induced immunomodulation, with increased survival of ()-infected hTg mice after a fatal LPS dose compared with naive mice or -infected hTg mice. Employing immunoprecipitation assays, hTg mice, and human immune cell culture, we demonstrate that hRetn binds the LPS receptor Toll-like receptor 4 (TLR4) through its N terminal and modulates STAT3 and TBK1 signaling, triggering a switch from proinflammatory to anti-inflammatory responses. Further, we generate hRetn N-terminal peptides that are able to block LPS proinflammatory function. Together, our studies identify a critical role for hRetn in blocking LPS function with important clinical significance in helminth-induced immunomodulation and sepsis.
“…Many studies show that tissue extracts or E/S products from helminths can suppress mammalian immune cell activation in vitro (29)(30)(31)(32) and ameliorate the severity of inflammatory disease in murine model systems (14,18,19,29,30,33,34). The demonstration herein that systemic administration of a crude extract of adult H. diminuta parasites dosedependently inhibits DSS-induced colitis adds to awareness of the ability of helminth-derived molecules to suppress intestinal inflammation (19,21,35).…”
Awareness of the immunological underpinnings of host-parasite interactions may reveal immune signaling pathways that could be used to treat inflammatory disease in humans. Previously we showed that infection with the rat tapeworm, Hymenolepis diminuta, used as a model helminth, or systemic delivery of worm antigen (HdAg) significantly reduced the severity of dinitrobenzene sulfonic acid (DNBS)-induced colitis in mice. Extending these analyses, intraperitoneal injection of HdAg dosedependently suppressed dextran sodium sulfate (DSS)-induced colitis, and this was paralleled by reduced gamma interferon (IFN-␥), interleukin-17 (IL-17), and tumor necrosis factor alpha (TNF-␣) production and increased IL-10 production from mitogen-activated splenocytes.
Until relatively recently, analysis of the host response to infection with helminth parasites focused almost invariably on TH2 immunity; however, it has emerged that helminth parasites trigger a complex regulatory network in their mammalian hosts that is characterized by cytokines (e.g., interleukin-10 [IL-10] and transforming growth factor  [TGF-]) and cellular components (e.g., regulatory macrophages and T cells) (1). Indeed, the development of an immunoregulatory environment likely contributes to the chronicity of helminth infection and asymptomatic disease. Moreover, individuals infected with a variety of species of helminths can be protected from concomitant disease as demonstrated in animal models of multiple sclerosis (2-4), joint (5-7) and gut (8-10) inflammation, and allergy (11, 12). In addition, treatment with somatic extracts or secreted products can significantly attenuate disease severity in models of inflammatory diseases (13-15), raising the possibility that isolation and purification of helminth-derived molecules could result in new anti-inflammatory drugs.The inverse relationship between the geographical distribution of inflammatory bowel disease (IBD) (i.e., Crohn's disease and ulcerative colitis) and areas of endemic helminth infection suggests that infection with helminth parasites may protect against IBD (16). Testing this hypothesis, infections with Trichinella spiralis, Schistosoma mansoni, and Heligmosomoides polygyrus were shown to inhibit inflammation in dinitrobenzene sulfonic acid (DNBS)-and dextran-sodium sulfate (DSS)-induced colitis and piroxicam-treated IL-10 Ϫ/Ϫ mice, respectively (8, 9, 17)-all established mouse models of colitis that share some similarities to human IBD. Similarly, and as an alternative to viable infection, systemic administration of helminth-derived antigens can ameliorate colitis in animal models. As examples, the excretory/secretory (E/S) products from adult T. spiralis reduced DSS-induced colitis (18) and S. mansoni egg antigens ameliorated immunemediated colitis (19): in both instances, suppression of TH1 and TH17 cytokines correlated with the beneficial anticolitic effect. While encouraging, the precise mechanism of action of any helminth-derived extract or molecule to block colitis or other inflammatory dis...
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