Lipopolysaccharide preconditioning augments phagocytosis of malaria-parasitized red blood cells by bone marrow-derived macrophages in the liver, thereby increasing the murine survival after Plasmodium yoelii infection

Malaria remains a grave concern for humans, as effective medical countermeasures for Plasmodium infection have yet to be developed. Phagocytic clearance of parasitized red blood cells (pRBCs) by macrophages is an important front-line innate host defense against Plasmodium infection. We previously showed that repeated injections of low-dose lipopolysaccharide (LPS) prior to bacterial infection, called LPS preconditioning, strongly augmented phagocytic/bactericidal activity in murine macrophages. However, if LPS preconditioning prevents murine Plasmodium infection is unclear. We investigated the protective effects of LPS preconditioning against lethal murine Plasmodium infection, focusing on CD11b high F4/80 low liver macrophages, which are increased by LPS preconditioning. Mice were subjected to LPS preconditioning by intraperitoneal injections of low-dose LPS for 3 consecutive days, and 24 h later, they were intravenously infected with pRBCs of Plasmodium yoelii 17XL. LPS preconditioning markedly increased the murine survival and reduced parasitemia, while it did not reduce TNF secretions, only delaying the peak of plasma IFN-γ after Plasmodium infection in mice. An in vitro phagocytic clearance assay of pRBCs showed that the CD11b high F4/80 low liver macrophages, but not spleen macrophages, in the LPS-preconditioned mice had significantly augmented phagocytic activity against pRBCs. The adoptive transfer of CD11b high F4/80 low liver macrophages from LPS-preconditioned mice to control mice significantly improved the survival after Plasmodium infection. We conclude that LPS preconditioning stimulated CD11b high F4/80 low liver macrophages to augment the phagocytic clearance of pRBCs, which may play a central role in resistance against Plasmodium infection.

Systemic administration of β-glucan induces immune training in microglia

Background An innate immune memory response can manifest in two ways: immune training and immune tolerance, which refers to an enhanced or suppressed immune response to a second challenge, respectively. Exposing monocytes to moderate-to-high amounts of bacterial lipopolysaccharide (LPS) induces immune tolerance, whereas fungal β-glucan (BG) induces immune training. In microglia, it has been shown that different LPS inocula in vivo can induce either immune training or tolerance. Few studies focused on impact of BG on microglia and were only performed in vitro. The aim of the current study was to determine whether BG activates and induces immune memory in microglia upon peripheral administration in vivo. Methods Two experimental designs were used. In the acute design, mice received an intraperitoneal (i.p.) injection with PBS, 1 mg/kg LPS or 20 mg/kg BG and were terminated after 3 h, 1 or 2 days. In the preconditioning design, animals were first challenged i.p. with PBS, 1 mg/kg LPS or 20 mg/kg BG. After 2, 7 or 14 days, mice received a second injection with PBS or 1 mg/kg LPS and were sacrificed 3 h later. Microglia were isolated by fluorescence-activated cell sorting, and cytokine gene expression levels were determined. In addition, a self-developed program was used to analyze microglia morphological changes. Cytokine concentrations in serum were determined by a cytokine array. Results Microglia exhibited a classical inflammatory response to LPS, showing significant upregulation of Tnf, Il6, Il1β, Ccl2, Ccl3 and Csf1 expression, three h after injection, and obvious morphological changes 1 and 2 days after injection. With an interval of 2 days between two challenges, both BG and LPS induced immune training in microglia. The training effect of LPS changed into immune tolerance after a 7-day interval between 2 LPS challenges. Preconditioning with BG and LPS resulted in increased morphological changes in microglia in response to a systemic LPS challenge compared to naïve microglia. Conclusions Our results demonstrate that preconditioning with BG and LPS both induced immune training of microglia at two days after the first challenge. However, with an interval of 7 days between the first and second challenge, LPS-preconditioning resulted in immune tolerance in microglia.

Systemic administration of β-glucan induces immune training in microglia

BackgroundAn innate immune memory response can manifest in two ways: immune training and immune tolerance, which refers to an enhanced or suppressed immune response to a second challenge, respectively. Exposing monocytes to moderate-to-high amounts of bacterial lipopolysaccharide (LPS) induces immune tolerance, whereas fungal β-glucan (BG) induces immune training. In microglia, it has been shown that different LPS inocula in vivo can induce either immune training or tolerance. Few studies focused on impact of BG on microglia and were only performed in vitro. The aim of the current study was to determine whether BG activates and induces immune memory in microglia upon peripheral administration in vivo.MethodsTwo experimental designs were used. In the acute design, mice received an intraperitoneal (i.p.) injection with PBS, 1 mg/kg LPS or 20 mg/kg BG and were terminated after 3 h, 1 or 2 days. In the preconditioning design, animals were first challenged i.p. with PBS, 1 mg/kg LPS or 20 mg/kg BG. After 2, 7 or 14 days, mice received a second injection with PBS or 1 mg/kg LPS and were sacrificed 3 h later. Microglia were isolated by fluorescence-activated cell sorting, and cytokine gene expression levels were determined. In addition, a self-developed program was used to analyze microglia morphological changes. Cytokine concentrations in serum were determined by a cytokine array.ResultsMicroglia exhibited a classical inflammatory response to LPS, showing significant upregulation of Tnf, Il6, Il1β, Ccl2, Ccl3 and Csf1 expression, three h after injection, and obvious morphological changes 1 and 2 days after injection. With an interval of 2 days between two challenges, both BG and LPS induced immune training in microglia. The training effect of LPS changed into immune tolerance after a 7-day interval between 2 LPS challenges. Preconditioning with BG and LPS resulted in increased morphological changes in microglia in response to a systemic LPS challenge compared to naïve microglia.ConclusionsOur results demonstrate that preconditioning with BG and LPS both induced immune training of microglia at two days after the first challenge. However, with an interval of 7 days between the first and second challenge, LPS-preconditioning resulted in immune tolerance in microglia.

Lipopolysaccharide preconditioning augments phagocytosis of malaria-parasitized red blood cells through induced bone marrow-derived macrophages in the liver, thereby increasing the murine survival after Plasmodium yoelii infection

Malaria remains a grave concern for humans, as effective medical countermeasures for malaria infection have yet to be developed. Phagocytic clearance of parasitized red blood cells (pRBCs) by macrophages is an important front-line innate host defense against malaria infection. We previously showed that repeated injections of low-dose lipopolysaccharide (LPS) prior to bacterial infection, called LPS preconditioning, strongly augmented phagocytic/bactericidal activity in murine macrophages. However, how LPS preconditioning prevents murine malaria infection is unclear. We investigated the protective effects of LPS preconditioning against lethal murine malaria infection, focusing on CD11bhigh F4/80low liver macrophages, which are increased by LPS preconditioning. Mice were subjected to LPS preconditioning by intraperitoneal injections of low-dose LPS for 3 consecutive days, and 24 h later, they were intravenously infected with pRBCs of Plasmodium yoelii 17XL. LPS preconditioning markedly increased the murine survival and reduced parasitemia, while it did not reduce TNF secretions, only delaying the peak of plasma IFN-γ after malaria infection in mice. An in vitro phagocytic clearance assay of pRBCs showed that the CD11bhigh F4/80low liver macrophages of the LPS-preconditioned mice had significantly augmented phagocytic activity against pRBCs. The adoptive transfer of CD11bhigh F4/80low liver macrophages from LPS-preconditioned mice to control mice significantly improved the survival after malaria infection. We conclude that LPS preconditioning stimulated CD11bhigh F4/80low liver macrophages to augment the phagocytic clearance of pRBCs, which may play a central role in resistance against malaria infection. LPS preconditioning may be an effective tool for preventing malaria infection.

Time-Dependent miRNA Profile in the Mouse Kidney during LPS Preconditioning

Background: Pre-treatment with lipopolysaccharide (LPS) protected the kidney against a later lethal ischemia. To reveal the mechanisms of renal cross-tolerance and septic acute kidney injury we investigated the effects of LPS on miRNA expression in the kidney. Methods: Male NMRI mice were injected with 40 and 10 mg/kg LPS ip. and sacrificed at 1.5 and 6 hours (early preconditioning, EP) and at 24 and 48 hours (late preconditioning, LP). The miRNA profile was established using miRCURY LNA™ microarray and confirmed with qPCR. Results: Plasma urea concentration peaked at 24 hours after LPS and decreased thereafter. Renal TNF-α and IL-6 mRNA were extremely elevated at all time-points. miRNome changes were mild at 1.5 hours, most miRNAs were altered at 6 and 24 hours and declined by 48 hours. Not all miRNAs could be assayed or validated by qPCR. In EP miR-762 was newly identified and validated and was the most elevated miRNA with both methods. In LP miR-21a-5p was the most influenced miRNA followed by miR-451a, miR-144-3p and miR-146a-5p. MiR-21a-3p increased significantly in both EP and LP. Conclusion: miR-762 might attenuate the LPS-induced immune response during EP and the miR-144/451 cluster is involved in LPS-induced renal preconditioning.

Endogenous protection from ischemic brain injury by preconditioned monocytes

Exposure to low dose lipopolysaccharide prior to cerebral ischemia is neuroprotective in stroke models, a phenomenon termed preconditioning. While it is well established that lipopolysaccharide-preconditioning induces central and peripheral immune responses, the cellular mechanisms modulating ischemic injury remain unclear. Here, we investigated the role of immune cells in the brain protection afforded by preconditioning and we tested whether monocytes may be reprogrammed by ex vivo lipopolysaccharide exposure thus modulating the inflammatory injury after cerebral ischemia in male mice. We found that systemic injection of low-dose lipopolysaccharide induces a distinct subclass of CD115+Ly6Chi monocytes that protect the brain after transient middle cerebral artery occlusion in mice. Remarkably, adoptive transfer of monocytes isolated from preconditioned mice into naïve mice 7 hours after transient middle cerebral artery occlusion reduced brain injury. Gene expression and functional studies showed that IL-10, iNOS and CCR2 in monocytes are essential for the neuroprotection. This protective activity was elicited even if mouse or human monocytes were exposed ex vivo to lipopolysaccharide and then injected into male mice after stroke. Cell tracking studies showed that protective monocytes are mobilized from the spleen and reach brain and meninges, wherein they suppressed post-ischemic inflammation and neutrophils influx into the brain parenchyma. Our findings unveil a previously unrecognized subpopulation of splenic monocytes capable to protect the brain with an extended therapeutic window, and provide the rationale for cell therapies based on the delivery of autologous or allogeneic protective monocytes into patients with ischemic stroke.Significance StatementInflammation is a key component of the pathophysiology of the brain in stroke, a leading cause of death and disability with limited therapeutic options. Here, we investigate endogenous mechanisms of protection against cerebral ischemia. Using LPS preconditioning as an approach to induce ischemic tolerance in mice, we found the generation of neuroprotective monocytes within the spleen from where they traffic to the brain and meninges suppressing post-ischemic inflammation. Importantly, systemic LPS preconditioning can be mimicked by adoptive transfer of in vitro-preconditioned mouse or human monocytes at translational relevant time points after stroke. This model of neuroprotection may facilitate clinical efforts to increase the efficacy of bone marrow mononuclear cell treatments in acute neurological diseases such as cerebral ischemia.