Dog hepatocytes are key effector cells in the liver innate immune response toLeishmania infantum

Abstract

Hepatocytes constitute the majority of hepatic cells, and play a key role in controlling systemic innate immunity, via pattern-recognition receptors (PRRs) and by synthesizing complement and acute phase proteins. Leishmania infantum, a protozoan parasite that causes human and canine leishmaniasis, infects liver by establishing inside the Kupffer cells. The current study proposes the elucidation of the immune response generated by dog hepatocytes when exposed to L. infantum. Additionally, the impact of adding leishmanicidal compound, meglumine antimoniate (MgA), to parasite-exposed hepatocytes was also addressed. L. infantum presents a high tropism to hepatocytes, establishing strong membrane interactions. The possibility of L. infantum internalization by hepatocytes was raised, but not confirmed. Hepatocytes were able to recognize parasite presence, inducing PRRs [nucleotide oligomerization domain (NOD)1, NOD2 and Toll-like receptor (TLR)2] gene expression and generating a mix pro- and anti-inflammatory cytokine response. Reduction of cytochrome P 450s enzyme activity was also observed concomitant with the inflammatory response. Addition of MgA increased NOD2, TLR4 and interleukin 10 gene expression, indicating an immunomodulatory role for MgA. Hepatocytes seem to have a major role in coordinating liver's innate immune response against L. infantum infection, activating inflammatory mechanisms, but always balancing the inflammatory response in order to avoid cell damage.

3D-Hepatocyte Culture Applied to Parasitology: Immune Activation of Canine Hepatic Spheroids Exposed to Leishmania infantum

“…In the current study, canine hepatic spheroids increased NO production in the presence of L. infantum , which was accompanied by the tendency to increase the expression of pro-inflammatory cytokines TNF-α and IL-6, indicating an immune recognition and activation of these cells towards the parasite. These findings are similar to what was previously observed by our group [ 24 ]. Interestingly, there are differences in the magnitude of the immune response to L. infantum generated by canine hepatic spheroids (3D) and traditional 2D cultured hepatocytes used in the previous study.…”

“…Canine ( Canis lupus familiaris , n = 10) hepatocytes were isolated by a two-step perfusion protocol followed by collagenase digestion and Percoll ® gradient centrifugation, as previously described [ 3 , 4 , 24 ]. Briefly, a liver lobule was cleaned of internal blood by perfusion, and tissue digestion was performed with collagenase H (Roche Diagnostics, Mannheim, Germany).…”

“…Gene expression of innate immune receptors (NOD1, NOD2, TLR2, TLR4, and TLR9) and cytokines (IL-10, IL-6, and TNF-α) were quantified by real-time polymerase chain reaction (PCR) quantitative method. The primers and protocols used were described by Rodrigues and co-workers [ 24 ]. Briefly, RNA extraction was performed using the NZY Total RNA Isolation Kit (Nzytech genes & enzymes, Lisboa, Portugal) and processed into cDNA synthesis, using NZY First-strand cDNA Synthesis Kit (Nzytech genes & enzymes) following the manufacturer’s indications.…”

“…Therefore, the present work constitutes a step forward from the previous study [ 24 ], as it takes advantage of the innovative 3D cell culture technique and applies it to canine hepatocytes, aiming to unravel the immune role played by dog hepatocytes when exposed to L. infantum parasites and the impact of inflammation on the metabolic activity of these cells.…”

Hepatocyte-derived IL-10 plays a crucial role in attenuating pathogenicity during the chronic phase of T. congolense infection

“…Moreover, we provide evidence that parasite-derived components, can trigger IL-10 production by hepatocytes from non-infected animals, indicating that they can sense and respond to the presence of trypanosomes. In this context, in another parasitic model, it was recently shown that hepatocytes can respond to Leishmania infantum infection by the activation of inflammatory mechanisms and the production of IL-10 in order to balance the inflammatory response and avoid cell damage [42].…”