IL-33 Enhances the Lipopolysaccharide-Induced Secretion of Inflammatory Cytokines and Ameliorates Lipopolysaccharide Desensitization in Macrophages

Background: Lipopolysaccharide (LPS) desensitization, which is characterized by hyporesponsiveness and a form of immunosuppression, is important in the negative regulation of responses to LPS and inflammatory disease such as sepsis. However, effect of IL-33 in the desensitization to LPS remains unclear. Methods: We used RNA-sequencing technology to analyze changes in mRNA in bone-marrow-derived macrophages (BMDMs) stimulated with LPS. Changes in expression and secretion of inflammatory cytokines were detected by qPCR and ELISA, respectively. Mechanisms were further studied through p65 phosphorylation detection. Results: IL-33 expression was significantly increased in LPS-treated macrophages, indicating its involvement in LPS-induced inflammation. Exogenous IL-33 increased the inflammatory response and ameliorated LPS desensitization by increasing the secretion of proinflammatory cytokines. It also activated p65 phosphorylation in resistant cells. Conclusion: IL-33 can enhance the inflammatory response induced by LPS and ameliorate LPS desensitization possibly by activating the NF-κB pathway in mouse macrophages.

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.

De novo synthesized polyunsaturated fatty acids operate as both host immunomodulators and nutrients for Mycobacterium tuberculosis

Successful control of Mycobacterium tuberculosis (Mtb) infection by macrophages relies on immunometabolic reprogramming, where the role of fatty acids (FAs) remains poorly understood. Recent studies unraveled Mtb's capacity to acquire saturated and monounsaturated FAs via the Mce1 importer. However upon activation, macrophages produce polyunsaturated FAs (PUFAs), mammal-specific FAs mediating the generation of immunomodulatory eicosanoids. Here, we asked how Mtb modulates de novo synthesis of PUFAs in primary mouse macrophages and whether this benefits host or pathogen. Quantitative lipidomics revealed that Mtb infection selectively activates the biosynthesis of w6 PUFAs upstream of the eicosanoid precursor arachidonic acid (AA), via transcriptional activation of Fads2. Inhibiting FADS2 in infected macrophages impaired their inflammatory and antimicrobial responses but had no effect on Mtb growth in mice. Using a click-chemistry approach, we found that Mtb efficiently imports w6 PUFAs via Mce1 in axenic culture, including AA. Further, Mtb preferentially internalized AA over all other FAs within infected macrophages, by mechanisms partially depending on Mce1 and supporting intracellular persistence. Notably, IFNγ repressed de novo synthesis of AA by infected mouse macrophages and restricted AA import by intracellular Mtb. Together, these findings identify AA as a major FA substrate for intracellular Mtb, whose mobilization by innate immune responses is opportunistically hijacked by the pathogen and downregulated by IFNγ.

SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-kB pathway

The pathogenesis of COVID-19 is associated with a hyperinflammatory response; however, the precise mechanism of SARS-CoV-2-induced inflammation is poorly understood. Here we investigated direct inflammatory functions of major structural proteins of SARS-CoV-2. We observed that spike (S) protein potently induced inflammatory cytokines and chemokines including IL-6, IL-1b, TNFa, CXCL1, CXCL2, and CCL2, but not IFNs in human and mouse macrophages. No such inflammatory response was observed in response to membrane (M), envelope (E), and nucleocapsid (N) proteins. When stimulated with extracellular S protein, human and mouse lung epithelial cells also produced inflammatory cytokines and chemokines. Interestingly, epithelial cells expressing S protein intracellularly were non-inflammatory, but elicited an inflammatory response in macrophages when co-cultured. Biochemical studies revealed that S protein triggers inflammation via activation of the NF-kB pathway in a MyD88-dependent manner. Further, such an activation of the NF-kB pathway was abrogated in Tlr2-deficient macrophages. Consistently, administration of S protein induced IL-6, TNF-a, and IL-1b in wild-type, but not Tlr2-deficient mice. Notably, upon recognition of S protein, TLR2 dimerizes with TLR1 or TLR6 to activate the NF-kB pathway. Together these data reveal a mechanism for the cytokine storm during SARS-CoV-2 infection and suggest that TLR2 could be a potential therapeutic target for COVID-19.

Resolvin T-series Reduce Neutrophil Extracellular Traps

The newly identified thirteen-series Resolvins (RvTs) regulate phagocyte functions and accelerate resolution of infectious inflammation. Since SARS-CoV-2 elicits uncontrolled inflammation involving neutrophil extracellular traps (NETs), we tested whether stereochemically defined RvTs regulate NET formation. Using microfluidic devices capturing NETs in PMA-stimulated human whole blood, the RvTs, RvT1-RvT4, 2.5 nM each, potently reduced NETs. With IL-1b-stimulated human neutrophils, each RvT dose- and time-dependently decreased NETosis giving ~50% potencies at 10 nM, compared to the known NETosis inhibitors [10 mM]. In mouse Staphylococcus aureus infection, RvTs [50 ng each] limited neutrophil infiltration, bacterial titers and NETs. Additionally, each RvT enhanced NET uptake by human macrophages; RvT2 was the most potent of the four RvTs, giving >50% increase in NET-phagocytosis. As part of the intracellular signaling mechanism, RvT2 increased cAMP and phospho-AMPK within human macrophages, and RvT2-stimulated NET uptake was abolished by PKA and AMPK inhibition. RvT2 also stimulated NET clearance by mouse macrophages in vivo. Together, these results provide evidence for novel pro-resolving functions of RvTs, namely reducing NETosis and enhancing macrophage NET clearance via a cAMP-PKA-AMPK axis. Thus, RvTs open opportunities for regulating NET-mediated collateral tissue damage during infection as well as monitoring NETs.

Bergamottin alleviates LPS-induced acute lung injury by inducing SIRT1 and suppressing NF-κB

Acute lung injury (ALI) is associated with a high mortality due to inflammatory cell infiltration and lung edema. The development of ALI commonly involves the activation of NF-κB. Since bergamottin is a natural furanocoumarin showing the ability to inhibit the activation of NF-κB, in this study we aimed to determine the effect of bergamottin on ALI. RAW264.7 mouse macrophages were pre-treated with bergamottin and then stimulated with LPS. Macrophage inflammatory responses were examined. Bergamottin (50 mg/kg body mass) was intraperitoneally administrated to mice 12 h before injection of LPS, and the effect of bergamottin on LPS-induced ALI was evaluated. Our results showed that LPS exposure led to increased production of TNF-α, IL-6, and monocyte chemoattractant protein-1 (MCP-1), which was impaired by bergamottin pre-treatment. In vivo studies confirmed that bergamottin pre-treatment suppressed LPS-induced lung inflammation and edema and reduced the levels of pro-inflammatory cytokines in lung tissues and bronchoalveolar lavage fluids. Mechanistically, bergamottin blocked LPS-induced activation of NF-κB signaling in lung tissues. Additionally, bergamottin treatment reduced NF-κB p65 protein acetylation, which was coupled with induction of SIRT1 expression. In conclusion, our results reveal the anti-inflammatory property of bergamottin in preventing ALI. Induction of SIRT1 and inhibition of NF-κB underlies the anti-inflammatory activity of bergamottin.