Lysosomal iron recycling in mouse macrophages is dependent upon both reductases LcytB and Steap3

Iron that is stored in macrophages as ferritin can be made bioavailable by degrading ferritin in the lysosome and releasing iron back into the cytosol. Iron stored in ferritin is found as Fe3+ and must be reduced to Fe2+ before it can be exported from the lysosome. Here we report that the lysosomal reductase Cyb561a3 (LcytB) and the endosomal reductase Six-transmembrane epithelial antigen of the prostate 3 (Steap3) act as lysosomal ferrireductases in the mouse macrophage cell line RAW264.7 converting Fe3+ to Fe2+ for iron recycling. We determined that when lysosomes were loaded with horse cationic ferritin, reductions or loss of LcytB or Steap3 using CrispR/Cas9-mediated knockout technology resulted in decreased lysosomal iron export. Loss of both reductases was additive in decreasing lysosomal iron export. Decreased reductase activity resulted in increased transcripts for iron acquisition proteins DMT1 and Tfrc1 suggesting cells were iron limited. We show transcript expression of LcytB and Steap3 is decreased in macrophages exposed to Escherichia coli pathogen UTI89 supporting a role for these reductases in regulating iron availability for pathogens. We further show that loss of LcytB and Steap3 in macrophages infected with UTI89, led to increased intracellular UTI89 proliferation suggesting that the endolysosomal system is retaining Fe3+ that can be used for intravesicular pathogen proliferation. Together, our findings reveal an important role for both LcytB and Steap3 in macrophage iron recycling and suggest that limiting iron recycling by decreasing expression of endolysosomal reductases is an innate immune response to protect against pathogen proliferation and sepsis.

The J2-Immortalized Murine Macrophage Cell Line Displays Phenotypical and Metabolic Features of Primary BMDMs in Their M1 and M2 Polarization State

Macrophages are immune cells that are important for the development of the defensive front line of the innate immune system. Following signal recognition, macrophages undergo activation toward specific functional states, consisting not only in the acquisition of specific features but also of peculiar metabolic programs associated with each function. For these reasons, macrophages are often isolated from mice to perform cellular assays to study the mechanisms mediating immune cell activation. This requires expensive and time-consuming breeding and housing of mice strains. To overcome this issue, we analyzed an in-house J2-generated immortalized macrophage cell line from BMDMs, both from a functional and metabolic point of view. By assaying the intracellular and extracellular metabolism coupled with the phenotypic features of immortalized versus primary BMDMs, we concluded that classically and alternatively immortalized macrophages display similar phenotypical, metabolic and functional features compared to primary cells polarized in the same way. Our study validates the use of this immortalized cell line as a suitable model with which to evaluate in vitro how perturbations can influence the phenotypical and functional features of murine macrophages.

Oxidative Stress Induced Cytotoxicity of Colloidal Copper Nanoparticles on RAW 264.7 Macrophage Cell Line

Copper is an essential trace mineral that plays an important role in various physiological processes of human body and also possesses excellent antimicrobial properties, however its high dose results in the formation of free-radicals, which can induce cytotoxicity through chromosomal and DNA damage. Therefore, cytotoxicity of colloidal copper nanoparticles (CuNPs) on murine macrophage cell line (RAW 264.7) was studied to understand the correlation between the cytotoxicity and the nanoparticle yield. Three identical sets of CuNPs with similar physical properties having hydrodynamic particle size of 11–14 nm were prepared by chemical reduction method with target yield of 0.2 g, 0.3 g and 0.4 g. CuNPs exhibited dose-dependent (0.001–100 μg/mL) cytotoxicity due to the mitochondrial damage as indicated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay. Oxidative stress induced by reactive oxygen species (ROS) in RAW 246.7 macrophage cell lines exposed to CuNPs was the primary cause of observed cytotoxicity in all CuNPs test samples. Morphological changes in cells also indicated strong dose-dependent oxidative damage by CuNPs. IC50 (half maximal inhibitory concentration) values of CuNPs were independent of nanoparticle yield. This suggests that per batch variation in CuNPs yield from 0.2 g to 0.4 g had no negative correlation with their toxicity that makes CuNPs a potential candidate for further development of nanotherapeutics and anticancer drugs.

Version up-date: The CDKN2B-AS1/ MIR497/TXNIP axis regulated macrophages

The polarization of macrophages plays a critical role in the pathophysiology of rheumatoid arthritis. The macrophages can have pro-inflammatory M1 polarization and various types of alternative anti-inflammatory M2 polarization. Our preliminary results showed that the CDKN2B-AS1/MIR497/TXNIP axis might regulate macrophages of rheumatoid arthritis patients. Therefore, we hypothesized that this axis regulated the polarization of rheumatoid macrophages. Flow cytometry was used to determine the surface polarization markers in M1 or M2 macrophages from healthy donors and rheumatoid arthritis patients. The QPCR and Western Blotting were used to compare the expression of the CDKN2B-AS1/MIR497/TXNIP axis in these macrophages. We Knocked down and overexpressed the axis in the macrophage cell line MD to test its roles in macrophage polarization. Compared to cells from healthy donors, cells from rheumatoid arthritis patients expressed higher levels of CD40 and CD80 and lower levels of CD16, CD163, CD206, and CD200R after polarization, they also expressed higher CDKN2B-AS1, lower MIR497, and higher TXNIP. In macrophages from healthy donors, there was no correlation among CDKN2B-AS1, MIR497, and TXNIP. But in macrophages from patients, there were significant correlations. The CDKN2B-AS1 knockdown, MIR497 mimics suppressed the M1 polarization but promoted the M2 polarization in MD cells, while the MIR497 knockdown and the TXNIP overexpression did the opposite. This study demonstrated that elevated CDKN2B-AS1 in macrophages promotes the M1 polarization and inhibited the M2 polarization of macrophages by the CDKN2B-AS1/ MIR497/TXNIP axis.

Expanding the Chemical Space of Aryloxy-naphthoquinones as Potential Anti-chagasic Agents: Synthesis and Trypanosomicidal Activity

In continuation our effort to research the chemical space of aryloxy-naphthoquinones as potential anti-Chagas agents, we synthesized nine derivatives and these compounds were evaluated in vitro against the epimastigote and trypomastigote forms of Mexican strains of Trypanosoma cruzi (T. cruzi). Most of these derivatives are highly active against epimastigote forms (IC50 < 1.0 µM) compared to the reference drug benznidazole (Bzn). Then these were evaluated on trypomastigotes, which is showing better potency results than Bzn for compounds 3b and 3g. In addition, the cytotoxicity of these compounds was determined on the murine macrophage cell line J774. 3b and 3i were the most selective compounds against NINOA trypomastigote and INC-5 epimastigote forms, respectively. Further these compounds also have good oral bioavailability according to theoretical predictions. Finally, we were able to determine optimal substitution patterns using pharmacophoric models. All these results are provided very useful structural information to continue our designing of naphthoquinone derivatives against T. cruzi.

New Aspects of Radiotherapy in Rheumatoid Arthritis Biomarker: Radio Sensitivity Response to Acute Ionizing Radiation Induces Cell Characteristic Reprogramming and Enhanced Cytokine Release of In Vitro Activated Macrophage Cell Line (RAW 264.7)

The response of biological systems to various types of radiations have many ambiguous dimensions. Among ionizing radiations, in vitro external gamma radiation therapy has mostly studied as model to declare the biological system challenges with radiation effects. Cell/organism exposure to gamma radiation, caused cascade of ionization events such as severe irreversible biological damages. However, the biological responses and oxidative stress related mechanisms under acute radiation conditions poorly understood in inflammatory systems. Following study tried to give a model about ionizing radiation effect on the macrophage that had a key role in inflammation mechanisms; to evaluate the impact of radiotherapy approach for inflammatory disease as rheumatoid arthritis. To this aim, Macrophage cell line (RAW 264.7) culture, exposed to different doses of gamma radiation (0,4, 6, 8, 10 Gy). Cell viability, apoptosis, cell cycle, migration; NO and PGE2 production; expression of pro-inflammatory and apoptotic genes and cytokine secretion of macrophages was also evaluated.The results showed that gamma treatment, at 4 Gy radiation, have slight effect on macrophage characteristics and cytokine secretion pattern. Versus, higher doses (8 and 10 Gy) increased DNA damage, expression of apoptotic genes and secretion of NO and PGE2 cytokines. 6 Gy radiation, the maximum radiation dose, show moderate nondestructive effects and inflammation process modulation. In this study, doses higher than 6 Gy of Gamma radiation caused cell mortality. It seems that 6 Gy Gamma radiation modulate the inflammatory cascade caused by macrophage cell, as a central core of autoimmune inflammatory disease in acute dose radiation therapy.

Novel azoles with potent antileishmanial activity

Aim: To investigate the antileishmanial activity of novel azole compounds against Leishmania donovani, which causes deadly visceral leishmaniasis disease. Materials & methods: A focused azole-based library was screened against both promastigotes and amastigotes forms of L. donovani strains in flat-bottomed 96-well tissue-culture plates and J774A.1 macrophage cell-line infected with L. donovani. The comprehensive screening of azole-based library against L. donovani strains provided novel hits, which can serve as a good starting point to initiate hit to lead optimization campaign. Results: Hits identified from azole-based library exhibited potent in vitro activity against promastigotes and amastigotes of L. donovani. Conclusion: These potent novel azole hits could be a good starting point to carry out for further medicinal chemistry exploration for antileishmania program.