Malarial pigment hemozoin (HZ) generates the lipoperoxidation product 4-hydroxynonenal (4-HNE), which is known to cause dysregulation of the immune response in malaria. The inhibition of granulocyte macrophage colony-stimulating factor (GM-CSF)-dependent differentiation of dendritic cells (DC) by HZ and 4-HNE was previously described in vitro, and the GM-CSF receptor (GM-CSF R) was hypothesised to be a primary target of 4-HNE in monocytes. In this study, we show the functional impact of HZ on GM-CSF R in monocytes and monocyte-derived DC by (i) impairing GM-CSF binding by 50 ± 9% and 65 ± 14%, respectively (n = 3 for both cell types); (ii) decreasing the expression of GM-CSF R functional subunit (CD116) on monocyte’s surface by 36 ± 11% (n = 6) and in cell lysate by 58 ± 16% (n = 3); and (iii) binding of 4-HNE to distinct amino acid residues on CD116. The data suggest that defective DC differentiation in malaria is caused by GM-CSF R dysregulation and GM-CSF R modification by lipoperoxidation product 4-HNE via direct interaction with its CD116 subunit.

Malaria is still the most important parasitic infectious disease. Numerous substances are known to have antimalarial activity; among them, artemisinin is the most widely used one, and artemisinin-based combination therapy (ACT) is recommended for the treatment of Plasmodium falciparum (P.f.) malaria. Antitumor, immunomodulatory, and other therapeutic applications of artemisinin are under extensive study. Several different mechanisms of action were proposed for dihydroartemisinin (DHA), the active metabolite of artemisinin, such as eliciting oxidative stress in target cells. The goal of this study is to monitor the generation of reactive oxygen species (ROS) and lipid peroxidation product 4-hydroxynonenal (4-HNE) by DHA in P.f.-infected human erythrocytes. Checking ROS and 4-HNE-protein adducts kinetics along the maturation of the parasite, we detected the highest level of 4-HNE in ring forms of P.f. due to DHA treatment. Low micromolar concentrations of DHA quickly induced levels of 4-HNE-adducts which are supposed to be damaging. Mass spectrometry identified the P.f. protein cysteine proteinase falcipain-1 as being heavily modified by 4-HNE, and plausibly, 4-HNE conjugation with vital P.f. proteins might contribute to DHA-elicited parasite death. In conclusion, significant 4-HNE accumulation was detectable after DHA treatment, though, at concentrations well above pharmacologically effective ranges in malaria treatment, but at concentrations described for antitumor activity. Thus, lipid peroxidation with consequent 4-HNE conjugation of functionally relevant proteins might be considered as a uniform mechanism for how DHA potentiates antimalarials’ action in ACT and controls the progression of tumors.

Malaria is a frequent parasitic infection becomes life threatening due to the disequilibrated immune responses of the host. Avid phagocytosis of malarial pigment hemozoin (HZ) and HZ-containing Plasmodium parasites incapacitates monocyte functions by bioactive lipoperoxidation products 4-hydroxynonenal (4-HNE) and hydroxyeicosatetraenoic acids (HETEs). CYP4F conjugation with 4-HNE is hypothesised to inhibit ω-hydroxylation of 15-HETE, leading to sustained monocyte dysfunction caused by 15-HETE accumulation. A combined immunochemical and mass-spectrometric approach identified 4-HNE-conjugated CYP4F11 in primary human HZ-laden and 4-HNE-treated monocytes. Six distinct 4-HNE-modified amino acid residues were revealed, of which C260 and H261 are localized in the substrate recognition site of CYP4F11. Functional consequences of enzyme modification were investigated on purified human CYP4F11. Palmitic acid, arachidonic acid, 12-HETE, and 15-HETE bound to unconjugated CYP4F11 with apparent dissociation constants of 52, 98, 38, and 73 µM, respectively, while in vitro conjugation with 4-HNE completely blocked substrate binding and enzymatic activity of CYP4F11. Gas chromatographic product profiles confirmed that unmodified CYP4F11 catalysed the ω-hydroxylation while 4-HNE-conjugated CYP4F11 did not. The 15-HETE dose dependently recapitulated the inhibition of the oxidative burst and dendritic cell differentiation by HZ. The inhibition of CYP4F11 by 4-HNE with consequent accumulation of 15-HETE is supposed to be a crucial step in immune suppression in monocytes and immune imbalance in malaria.

Objective: Malaria remains an important life-threatening disease that provokes a complex immune response, marked by an initial immune boost followed by long-term suppression, warranting further study. One of the manifestations of compromised immune response is co-infections, which are frequently reported in malaria patients and post-malaria convalescent individuals. Monocyte motility is a crucial step in immune cell recruitment, but this process is not fully efficient in malaria. Malarial pigment hemozoin, avidly phagocytosed by monocytes, inhibits important monocyte functions through lipoperoxidation. Methods: 4-Hydroxynonenal (4-HNE) adducts were detected on primary human monocytes by fluorescent microscopy, and CCR2 and TNFR1/2 receptors by flow cytometry. A two-dimensional migration microfluidic assay was applied for studying monocyte motility. Results: We show here that, following hemozoin ingestion, monocyte motility is inhibited. This is accompanied by a 2.4 ± 0.3-fold increase of the adducts of the lipoperoxidation product 4-HNE with monocyte proteins. Reduction of cell directional motility by 3.2 ± 0.6 times in response to MCP-1 and by 3.8 ± 1.1 times in response to TNF-α is observed alongside a decrease in CCR2 expression by 55 ± 8%, TNFR1 (CD120a) expression by 79 ± 4% and TNFR2 (CD120b) expression by 58 ± 7%. Conclusions: The low availability and potential malfunction of these important chemotactic receptors could be proposed as an additional mechanism for the poor immune response in malaria. Therapeutic relevance could be found in interventions aimed at regulating damaged or downregulated receptors and the application of antioxidants or other reagents to contrast protein addition of free 4-HNE. Significance statement: The study deepened our knowledge about lipid peroxidation processes, which are related to infectious diseases and immunity. Observed impaired immune motility and collapse of receptors could explain immunosuppression manifestations and co-infections in malaria and other diseases accompanied by oxidative stress.

Visceral fat mass is associated with a condition of chronic inflammation and can predispose the overweight to an increased cancer risk. Although it is known that adipocytes are active producers of the pro-inflammatory transforming growth factor β (TGFβ), the causes of their excessive synthesis are not clear. In this study, we reproduced two metabolic stress conditions frequently occurring in vivo, namely hypoxia and the fatty acid-driven metabolic uncoupling, and we characterized the response of an in vitro model of 3T3-L1 mouse adipocytes. For the first time, we demonstrated that the mitochondrial dysmetabolism of differentiated adipocytes induced the secretion of TGFβ. The paracrine activity of the secreted cytokine was then tested on two human pancreatic cancer cell lines. Cancer cells responded to the stimulation by increasing mitochondrial respiration, switching on the epithelial–mesenchymal transition (EMT) program and enhancing their motility. The data obtained in this proof-of-concept research show that TGFβ can be produced by dysmetabolic adipocytes, linking the altered metabolism with pro-tumorigenic inflammation. The novel observations of this study identify in metabolic stress a still unexplored cause of inflammation and cancer progression and pave the way to more detailed in vitro and clinical studies on pancreatic cancer.