TLR-dependent hepcidin expression by THP-1 cells in response to TLR4 and TLR9 (LPS and CpG)
Al-Lawati, Hiba Abdul Redha
Cardiff Metropolitan University
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Linking immunity with iron can be well illustrated by the production of hepcidin as it is considered to be the master regulator of iron metabolism in mammals. Iron is an essential element for microbes and higher animals and successful microbial pathogens have evolved multiple mechanisms to selectively acquire iron from the host. As a counter-measure, the host immune system employs a variety of mechanisms to withhold iron from microbial invaders to prevent or control infections. Notably among these mechanisms is the production of hepcidin, which is known as an antimicrobial peptide. It has been demonstrated that mouse macrophages and neutrophils produce hepcidin upon bacterial challenge. This production depends on the activation of Toll-like receptor (TLR) 4, a member of the TLR family of receptors, which is critically involved in microbial recognition. Little is known about the capacity of myeloid cells at different stages of differentiation to produce hepcidin. Furthermore, limited information is available regarding whether stimulation of other TLRs than TLR4 can also result in hepcidin production. Therefore, it was the main aim of this work to assess the capacity of the human monocytic cell line THP-I before or after differentiation to produce hepcidin constitutively or upon TLR-mediated activation by TLR3-, TLR4- and TLR9- specific ligands. The outcome of this project is that if THP-I cells can be differentiated and activated by PMA, they are able to respond to stimulation via TLR4 and via TLR9 using the specific ligands LPS and CpG by the production of IL-8, IL-6 and TNF-α cytokines. Though, this combination of ligands does not result in hepcidin expression. Of significance, the only combination of ligands that resulted in hepcidin expression was CpG/Poly I:C suggesting a putative role for hepcidin during dual infections with viral and bacterial pathogens as CpG and Poly I:C, which mimic these types of infection. recycling of senescent erythrocytes. When iron stores are low, hepcidin production is suppressed and ferroportin molecules are displayed, thus allowing iron to be released into the circulation from enterocytes and macrophages.7 Although the role of hepcidin in iron metabolism is well documented, its putative capacity to influence the course of the innate immune response has not been investigated in detail. It has been demonstrated that mouse macrophages and neutrophils produce hepcidin upon bacterial challenge.2OThis production depends on the activation of Toll-like receptor (TLR) 4, a member of the TLR family of receptors, which is critically involved in microbial recognition. Toll receptor wase first described in insects (fruit fly) in which this receptor plays a key role in protection against microbial infections.l3 In mammalians, TLRs are one of the most important receptors of the innate immune system comprising a family of at least 13 different membrane-associated molecules that can recognize different classes of pathogen associated molecular patterns (PAMPs). TLRs are found on many cells including epithelial cells and cells of the innate immune system including macrophages, dendritic cells (DCs) and B cells. TLR4 is the most thoroughly studied TLR, because of its outstanding role in antibacterial defence and its peculiar modes of signal transduction. It is well established that lipopolysaccharide (LPS) from Gram-negative bacteria is the main ligand for TLR4; even though an effective immune response to LPS requires numerous additional players. LPS from bacteria, such as Salmonella has long been known to induce a reaction in the infected host, and systemic injection of LPS into an animal causes a collapse of the circulatory and respiratory systems, a condition known as a shock.14 The role of other TLRs in hepcidin production and the physiological relevance of locally-produced hepcidin during infection and inflammation have escaped attention thus far. Furthermore, it is not known whether other more immature myeloid cells including monocytic cell lines have the capacity to produce hepcidin constitutively or upon microbial challenge. Therefore, it was the main aim of this work to assess the capacity of the human monocytic cell line THP-I to produce hepcidin constitutively or upon TLR-mediated activation. This cell line has been extensively used as an in vitro model of human monocytes, and its phenotype resembles that of an immature cell. Differentiation of THP-1 cells induced with phorbol esters or calcitriol results in cells with a more mature phenotype resembling macrophages. Hepcidin expression, at the mRNA level, was tested by using RT-PCR in undifferentiated and differentiated THP-I cells before and after treatment with the TLR4- and TlR9-specific ligands LPS and CpG DNA, respectively. These TLR ligands were used separately or in combination for cell stimulation. Furthermore, in order to confirm that THP-I cells were susceptible to stimulation with these agonist, the levels of pro-inflammatory cytokines (IL-6; IL-8 and TNF-α) in the culture supernatants were also measured. Of relevance the only combination of ligands that resulted in hepcidin expression was CpG/Poly I:C suggesting a putative role for hepcidin during dual infections with viral and bacterial pathogens as CpG and Poly I:C mimic these types of infection.
BSc (Hons) Biomedical Science
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