Microglia depletion and repopulation do not alter the effects of cranial irradiation on hippocampal neurogenesis.

Cranial radiotherapy can cause lifelong cognitive complications in childhood brain tumor survivors, and reduced hippocampal neurogenesis is hypothesized to contribute to this. Following irradiation (IR), microglia clear dead neural progenitors and give rise to a neuroinflammatory microenvironment, which promotes a switch in surviving progenitors from neuronal to glial differentiation. Recently, depletion and repopulation of microglia were shown to promote neurogenesis and ameliorate cognitive deficits in various brain injury models. In this study, we utilized the Cx3cr1CreERt2-YFP/+Rosa26DTA/+ transgenic mouse model to deplete microglia in the juvenile mouse brain before subjecting them to whole-brain IR and investigated the short- and long-term effects on hippocampal neurogenesis. Within the initial 24 h after IR, the absence of microglia led to an accumulation of dead cells in the subgranular zone, and 50-fold higher levels of the chemokine C-C motif ligand 2 (CCL2), in sham brains and 7-fold higher levels after IR. The absence of microglia, and the subsequent repopulation within 10 days, did neither affect the loss of proliferating or doublecortin-positive cells, nor the reduced growth of the granule cell layer. Our results argue against a role for a pro-inflammatory microenvironment in the dysregulation of hippocampal neurogenesis and suggest that the observed reduction of neurogenesis was solely due to IR.

Novel recombinant vaccinia virus-vectored vaccine affords complete protection against homologous Borrelia burgdorferi infection in mice.

The rising prevalence of Lyme disease (LD) in North America and Europe has emerged as a pressing public health concern. Despite the availability of veterinary LD vaccines, no vaccine is currently available for human use. Outer surface protein C (OspC) found on the outer membrane of the causative agent, Borrelia burgdorferi, has been identified as a promising target for LD vaccine development due to its sustained expression during mammalian infection. However, the efficacy and immunological mechanisms of LD vaccines solely targeting OspC are not well characterized. In this study, we developed an attenuated Vaccinia virus (VV) vectored vaccine encoding type A OspC (VV-OspC-A). Two doses of the VV-OspC-A vaccine conferred complete protection against homologous B. burgdorferi challenge in mice. Furthermore, the candidate vaccine also prevented the development of carditis and lymph node hyperplasia associated with LD. When investigating the humoral immune response to vaccination, VV-OspC-A was found to induce a robust antibody response predominated by the IgG2a subtype, indicating a Th1-bias. Using a novel quantitative flow cytometry assay, we also determined that elicited antibodies were capable of inducing antibody-dependent cellular phagocytosis in vitro. Finally, we demonstrated that VV-OspC-A vaccination generated a strong antigen-specific CD4+ T-cell response characterized by the secretion of numerous cytokines upon stimulation of splenocytes with OspC peptides. This study suggests a promising avenue for LD vaccine development utilizing viral vectors targeting OspC and provides insights into the immunological mechanisms that confer protection against B. burgdorferi infection.

Macrophage membrane coated discoidal polymeric particles for evading phagocytosis.

The purpose of this study was to investigate the potential of discoidal polymeric particles (DPPs) coated with macrophage membranes as a novel drug delivery system. The study aimed to determine whether these coated particles could reduce phagocytosis, and target specific organs, thereby enhancing drug delivery efficacy. In this study, discoidal polymeric particles (DPPs) were synthesized by a top-down fabrication method serving as the core drug delivery platform. The method involved the fusion of macrophage cell membrane vesicles with DPPs, resulting in macrophage membrane coated DPPs. This process aimed to translocate membrane proteins from macrophages onto the DPPs, rendering them structurally and functionally like host cells. The results of this study showed that macrophage membrane coated DPPs exhibited a threefold reduction in phagocytosis compared to bare DPPs. This reduction in phagocytosis indicated the potential of these coated DPPs to evade immune clearance. Time-lapse microscopy further illustrated the distinct interactions of macrophage membrane coated DPPs with immune cells. Biodistribution studies revealed that these coated particles displayed preferential accumulation in the lungs at early time points, followed by sustained accumulation in the liver. In conclusion, this study demonstrated that macrophage membrane coated DPPs represent a unique and promising strategy for drug delivery. These particles can mimic cell surfaces, reduce phagocytosis, and target specific organs. This opens exciting avenues for improving drug delivery efficacy in diverse therapeutic contexts. These findings advance our understanding of nanomedicine's potential in personalized therapies and targeted drug delivery strategies. Supplementary Information: The online version contains supplementary material available at 10.1007/s13534-024-00396-x.

Heterogeneity of Salmonella enterica lipopolysaccharide counteracts macrophage and antimicrobial peptide defenses.

Salmonella enterica is comprised of over 2,500 serovars, in which non-typhoidal serovars (NTS), Enteritidis (SE), and Typhimurium (STM) are the most clinically associated with human infections. Although NTS have similar genetic elements to cause disease, phenotypic variation including differences in lipopolysaccharide (LPS) composition may control immune evasion. Here, we demonstrate that macrophage host defenses and LL-37 antimicrobial efficacy against SE and STM are substantially altered by LPS heterogeneity. We found that SE evades macrophage killing by inhibiting phagocytosis while STM survives better intracellularly post-phagocytosis. SE-infected macrophages failed to activate the inflammasomes and subsequently produced less interleukin-1ß (IL-1ß), IL-18, and interferon λ. Inactivation of LPS biosynthesis genes altered LPS composition, and the SE LPS-altered mutants could no longer inhibit phagocytosis, inflammasome activation, and type II interferon signaling. In addition, SE and STM showed differential susceptibility to the antimicrobials LL-37 and colistin, and alteration of LPS structure substantially increased susceptibility to these molecules. Collectively, our findings highlight that modification of LPS composition by Salmonella increases resistance to host defenses and antibiotics.

Giant viruses inhibit superinfection by downregulating phagocytosis in Acanthamoeba.

In the context of the virosphere, viral particles can compete for host cells. In this scenario, some viruses block the entry of exogenous virions upon infecting a cell, a phenomenon known as superinfection inhibition. The molecular mechanisms associated with superinfection inhibition vary depending on the viral species and the host, but generally, blocking superinfection ensures the genetic supremacy of the virus's progeny that first infects the cell. Giant amoeba-infecting viruses have attracted the scientific community's attention due to the complexity of their particles and genomes. However, there are no studies on the occurrence of superinfection and its inhibition induced by giant viruses. This study shows that mimivirus, moumouvirus, and megavirus, exhibit different strategies related to the infection of Acanthamoeba. For the first time, we have reported that mimivirus and moumouvirus induce superinfection inhibition in amoebas. Interestingly, megaviruses do not exhibit this ability, allowing continuous entry of exogenous virions into infected amoebas. Our investigation into the mechanisms behind superinfection blockage reveals that mimivirus and moumouvirus inhibit amoebic phagocytosis, leading to significant changes in the morphology and activity of the host cells. In contrast, megavirus-infected amoebas continue incorporating newly formed virions, negatively affecting the available viral progeny. This effect, however, is reversible with chemical inhibition of phagocytosis. This work contributes to the understanding of superinfection and its inhibition in mimivirus, moumouvirus, and megavirus, demonstrating that despite their evolutionary relatedness, these viruses exhibit profound differences in their interactions with their hosts.IMPORTANCESome viruses block the entry of new virions upon infecting a cell, a phenomenon known as superinfection inhibition. Superinfection inhibition in giant viruses has yet to be studied. This study reveals that even closely related viruses, such as mimivirus, moumouvirus, and megavirus, have different infection strategies for Acanthamoeba. For the first time, we have reported that mimivirus and moumouvirus induce superinfection inhibition in amoebas. In contrast, megaviruses do not exhibit this ability, allowing continuous entry of exogenous virions into infected amoebas. Our investigation shows that mimivirus and moumouvirus inhibit amoebic phagocytosis, causing significant changes in host cell morphology and activity. Megavirus-infected amoebas, however, continue incorporating newly formed viruses, affecting viral progeny. This research enhances our understanding of superinfection inhibition in these viruses, highlighting their differences in host interactions.

Microglial Phagocytosis During Embryonic and Postnatal Development.

Microglia play decisive roles during the development of the central nervous system (CNS). Phagocytosis is one of the classical functions attributed to microglia, being involved in nearly all phases of the embryonic and postnatal development of the brain, such as rapid clearance of cell debris to avoid an inflammatory response, controlling the number of neuronal and glial cells or their precursors, contribution to axon guidance and to refinement of synaptic connections. To carry out all these tasks, microglial cells are equipped with a panoply of receptors, that convert microglia to the "professional phagocytes" of the nervous parenchyma. These receptors are modulated by spatiotemporal cues that adapt the properties of microglia to the needs of the developing CNS. Thus, in this chapter, we will discuss the role of microglial phagocytosis in all the aforementioned processes. First, we will explain the general phagocytic process, to describe afterward the performance of microglial cells in detail.

Synapse Regulation.

Microglia are the resident immune cells of the brain. As such, they rapidly detect changes in normal brain homeostasis and accurately respond by fine-tuning in a tightly regulated manner their morphology, gene expression, and functional behavior. Depending on the nature of these changes, microglia can thicken and retract their processes, proliferate and migrate, release numerous signaling factors and compounds influencing neuronal physiology (e.g., cytokines and trophic factors), in addition to secreting proteases able to transform the extracellular matrix, and phagocytosing various types of cellular debris, etc. Because microglia also transform rapidly (on a time scale of minutes) during experimental procedures, studying these very special cells requires methods that are specifically non-invasive. The development of such methods has provided unprecedented insights into the roles of microglia during normal physiological conditions. In particular, transcranial two-photon in vivo imaging revealed that presumably "resting" microglia continuously survey the brain parenchyma with their highly motile processes, in addition to modulating their structural and functional interactions with neuronal circuits along the changes in neuronal activity and behavioral experience occurring throughout the lifespan. In this chapter, we will describe how surveillant microglia interact with synaptic elements and modulate the number, maturation, function, and plasticity of synapses in the healthy developing, mature, and aging brain, with consequences on neuronal activity, learning and memory, and the behavioral outcome.

Microglia and Systemic Immunity.

Microglia are specialized immune cells that reside in the central nervous system (CNS) and play a crucial role in maintaining the homeostasis of the brain microenvironment. While traditionally regarded as a part of the innate immune system, recent research has highlighted their role in adaptive immunity. The CNS is no longer considered an immune-privileged organ, and increasing evidence suggests bidirectional communication between the immune system and the CNS. Microglia are sensitive to systemic immune signals and can respond to systemic inflammation by producing various inflammatory cytokines and chemokines. This response is mediated by activating pattern recognition receptors (PRRs), which recognize pathogen- and danger-associated molecular patterns in the systemic circulation. The microglial response to systemic inflammation has been implicated in several neurological conditions, including depression, anxiety, and cognitive impairment. Understanding the complex interplay between microglia and systemic immunity is crucial for developing therapeutic interventions to modulate immune responses in the CNS.

Roles in Innate Immunity.

Microglia are best known as the resident phagocytes of the central nervous system (CNS). As a resident brain immune cell population, microglia play key roles during the initiation, propagation, and resolution of inflammation. The discovery of resident adaptive immune cells in the CNS has unveiled a relationship between microglia and adaptive immune cells for CNS immune-surveillance during health and disease. The interaction of microglia with elements of the peripheral immune system and other CNS resident cells mediates a fine balance between neuroprotection and tissue damage. In this chapter, we highlight the innate immune properties of microglia, with a focus on how pattern recognition receptors, inflammatory signaling cascades, phagocytosis, and the interaction between microglia and adaptive immune cells regulate events that initiate an inflammatory or neuroprotective response within the CNS that modulates immune-mediated disease exacerbation or resolution.

Role of Microglia in Stroke.

Ischemic stroke is a complex brain pathology caused by an interruption of blood supply to the brain. It results in neurological deficits which that reflect the localization and the size of the compromised brain area and are the manifestation of complex pathogenic events triggered by energy depletion. Inflammation plays a prominent role, worsening the injury in the early phase and influencing poststroke recovery in the late phase. Activated microglia are one of the most important cellular components of poststroke inflammation, appearing from the first few hours and persisting for days and weeks after stroke injury. In this chapter, we will discuss the nature of the inflammatory response in brain ischemia, the contribution of microglia to injury and regeneration after stroke, and finally, how ischemic stroke directly affects microglia functions and survival.

Deletion of the WD40 domain of ATG16L1 exacerbates acute pancreatitis, abolishes LAP-like non-canonical autophagy and slows trypsin degradation.

The WD40 domain (WDD) of ATG16L1 plays a pivotal role in non-canonical autophagy. This study examined the role of recently identified LAP-like non-canonical autophagy (LNCA) in acute pancreatitis. LNCA involves rapid single-membrane LC3 conjugation to endocytic vacuoles in pancreatic acinar cells. The rationale for this study was the previously observed presence of trypsin in the organelles undergoing LNCA; aberrant trypsin formation is an important factor in pancreatitis development. Here we report that the deletion of WDD (attained in ATG16L1[E230] mice) eliminated LNCA, aggravated caerulein-induced acute pancreatitis and suppressed the fast trypsin degradation observed in both a rapid caerulein-induced disease model and in caerulein-treated isolated pancreatic acinar cells. These experiments indicate that LNCA is a WDD-dependent mechanism and suggest that it plays not an activating but a protective role in acute pancreatitis. Furthermore, palmitoleic acid, another inducer of experimental acute pancreatitis, strongly inhibited LNCA, suggesting a novel mechanism of pancreatic lipotoxicity.Abbreviation: AMY: amylase; AP: acute pancreatitis; CASM: conjugation of Atg8 to single membranes; CCK: cholecystokinin; FAEE model: fatty acid and ethanol model; IL6: interleukin 6; LA: linoleic acid; LAP: LC3-associated phagocytosis; LMPO: lung myeloperoxidase; LNCA: LAP-like non-canonical autophagy; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MPO: myeloperoxidase; PMPO: pancreatic myeloperoxidase; POA: palmitoleic acid; WDD: WD40 domain; WT: wild type.

Godanti bhasma (anhydrous CaSO4) induces massive cytoplasmic vacuolation in mammalian cells: A model for phagocytosis assay.

Phagocytosis is an essential physiological mechanism; its impairment is associated with many diseases. A highly smart particle is required for understanding detailed sequential cellular events in phagocytosis. Recently, we identified an Indian traditional medicine named Godanti Bhasma (GB), a bioactive calcium sulfate particle prepared by thermo-transformation ofgypsum. Thermal processing of the gypsum transforms its native physicochemical properties by removing water molecules into the anhydrous GB, which was confirmed by Raman and FT-IR spectroscopy. GB particle showed a 0.5-5 µm size range and a neutral surface charge. Exposure of mammalian cells to GB particles showed a rapid cellular uptake through phagocytosis and induced massive cytoplasmic vacuolation in cells. Interestingly, no cellular uptake and cytoplasmic vacuolation were observed with the parent gypsum particle. The presence of the GB particles in intra-vacuolar space was confirmed using FESEM coupled with EDX. Flow cytometry analysis and live tracking of GB-treated cells showed particle internalization, vacuole formation, particle dissolution, and later vacuolar turnover. Quantification of GB-induced vacuolation was done using neutral red uptake assay in cells. Treatment of lysosomal inhibitors (BFA1 or CQ) with GB could not induce vacuolation, suggesting the requirement of an acidic environment for the vacuolation. In the mimicking experiment, GB particle dissolution in acidic cell-free solution suggested that degradation of GB occurs by acidic pH inside the cell vacuole. Vacuole formation generally accompanies with cell death, whereas GB-induced massive vacuolation does not cause cell death. Moreover, the cell divides and proliferates with the vacuolar process, intra-vacuolar cargo degradation, and eventually vacuolar turnover. Taken together, the sequential cellular events in this study suggest that GB can be used as a smart particle for phagocytosis assay development in animal cells.

Antimicrobial and immunomodulatory activities of porcine cathelicidin Protegrin-1.

Antimicrobial peptides (AMPs) are a promising alternative to antibiotics in the fight against multi-drug resistant and immune system-evading bacterial infections. Protegrins are porcine cathelicidins which have been identified in porcine leukocytes. Protegrin-1 is the best characterized family member and has broad antibacterial activity by interacting and permeabilizing bacterial membranes. Many host defense peptides (HDPs) like LL-37 or chicken cathelicidin 2 (CATH-2) have also been shown to have protective biological functions during infections. In this regard, it is interesting to study if Protegrin-1 has the immune modulating potential to suppress unnecessary immune activation by neutralizing endotoxins or by influencing the macrophage functionality in addition to its direct antimicrobial properties. This study showed that Protegrin-1 neutralized lipopolysaccharide- (LPS) and bacteria-induced activation of RAW macrophages by binding and preventing LPS from cell surface attachment. Furthermore, the peptide treatment not only inhibited bacterial phagocytosis by murine and porcine macrophages but also interfered with cell surface and intracellular bacterial survival. Lastly, Protegrin-1 pre-treatment was shown to inhibit the amastigote survival in Leishmania infected macrophages. These experiments describe an extended potential of Protegrin-1's protective role during microbial infections and add to the research towards clinical application of cationic AMPs.

CpG oligonucleotides induce an acute murine thrombocytopenia dependent on toll-like receptor 9 and spleen tyrosine kinase pathways.

BACKGROUND: CpG ODN are synthetic single stranded DNA sequences that act as immunostimulants. They have been increasingly used to treat several cancers, however, thrombocytopenia is a potential recognized side effect of some sequences. OBJECTIVES: We tested the ability of two CpG ODN (ODN 2395 and ISIS 120704) to induce thrombocytopenia when administered to BALB/c mice and determined mechanisms associated with thrombocytopenia. METHODS: BALB/c mice were pre-bled and then injected with titrated doses of CpG ODNs and platelet counts were determined. The mice were treated IVIg or with various inhibitors and antagonists of toll-like receptor 9 (TLR9) and spleen tyrosine kinase (Syk) to determine their effects on the thrombocytopenia. RESULTS AND CONCLUSIONS: Compared with saline-treated mice or mice treated with 2'-O-methoxyethyl (MOE)-modified antisense (ASO) ODN, both ODN 2395 and ISIS 120704 induced an acute dose-dependent thrombocytopenia within 3 and 24 hours respectively. The thrombocytopenia was associated with significant increases in plasma monocyte chemoattractant protein 1 (MCP1). Intravenous immunoglobulin (IVIg) administration significantly rescued the CpG ODN-induced thrombocytopenia, as did treatment with either a Syk-inhibitor or TLR9 antagonists. In vitro, CpG ODN could activate human platelets and this correlated significantly with enhanced IVIg- and Syk-dependent phagocytosis by THP-1 monocytes. These results suggest that CpG ODN induce an acute inflammatory-associated (IVIg-sensitive) thrombocytopenia that can be alleviated by Syk- or TLR9-blockade, and an IVIg- and Syk-dependent platelet clearance pathway appears primarily responsible for the thrombocytopenia. Whether these results are applicable to humans still has to be elucidated.

The ability of human TIM1 to bind phosphatidylethanolamine enhances viral uptake and efferocytosis compared to rhesus and mouse orthologs.

T-cell Immunoglobulin and Mucin (TIM)-family proteins facilitate the clearance of apoptotic cells, are involved in immune regulation, and promote infection of enveloped viruses. These processes are frequently studied in experimental animals such as mice or rhesus macaques, but functional differences among the TIM orthologs from these species have not been described. Previously, we reported that while all three human TIM proteins bind phosphatidylserine (PS), only human TIM1 (hTIM1) binds phosphatidylethanolamine (PE), and that this PE-binding ability contributes to both phagocytic clearance of apoptotic cells and virus infection. Here we show that rhesus macaque TIM1 (rhTIM1) and mouse TIM1 (mTIM1) bind PS but not PE and that their inability to bind PE makes them less efficient than hTIM1. We also show that alteration of only two residues of mTIM1 or rhTIM1 enables them to bind both PE and PS, and that these PE-binding variants are more efficient at phagocytosis and mediating viral entry. Further, we demonstrate that the mucin domain also contributes to the binding of the virions and apoptotic cells, although it does not directly bind phospholipid. Interestingly, contribution of the hTIM1 mucin domain is more pronounced in the presence of a PE-binding head domain. These results demonstrate that rhTIM1 and mTIM1 are inherently less functional than hTIM1, owing to their inability to bind PE and their less functional mucin domains. They also imply that mouse and macaque models underestimate the activity of hTIM1.

Cell-in-cell phenomena of intracellular neutrophils in a recurrent pleomorphic xanthoastrocytoma.

We describe a case of a young patient with a recurrent pleomorphic xanthoastrocytoma (PXA) showing unusual cell-in-cell (CiC) phenomena. We observed mostly viable but also necrotic neutrophils engulfed within tumor cells. The recurrent tumor was immunopositive for BRAFV600E mutant protein and showed CDKN2 homozygous deletions typical of PXA. Both genetic alterations were also reported in the original primary tumor. Unlike the original tumor that was GFAP and Olig-2 immunopositive, the recurrent neoplasm was largely negative for GFAP and Olig-2 suggesting dedifferentiation. The large malignant cells that contained the neutrophils were negative for histiocytic and lymphohematopoietic markers. Whereas CDKN2 homozygous deletion is common in PXA, its presence is rare in histiocytic neoplasms. Both reactive astrocytes and glial neoplasms very rarely may engulf neutrophils in a process resembling emperipolesis or cellular cannibalism. Future work may clarify which type of CiC pathway is involved.

Characterization of integrins in cellular immunity of the oriental armyworm, Mythimna separata.

Insect hemocytes eliminate foreign substances from the hemocoel through various immune reactions. Integrins, receptor proteins present on the cell membrane, are formed as a heterodimer from α and ß subunits and are known to be involved in various immune reactions. To elucidate the role of integrins in the immunity of the lepidoptera Mythimna separata, genes encoding integrins were screened from the genome, resulting in the identification of eight α and four ß integrin genes. The expression levels of the integrin genes did not change in response to the injection of small abiotic beads undergoing phagocytosis in M. separata larvae. However, significant inductions of some integrin gene expressions were observed in hemocytes that formed capsules around large abiotic beads during encapsulation, especially in MysIntα2. Under biotic stimulation, induction of the MysIntα2 was evident after exposures to Gram-negative bacteria (Escherichia coli) and entomopathogenic nematodes (Steinernema carpocapsae), but not to Gram-positive bacteria (Micrococcus luteus). Immunostaining analysis revealed that MysIntα2 was specifically localized to hemocytes surrounding the beads during the encapsulation reaction. Furthermore, the spreading and encapsulation abilities of hemocytes were significantly inhibited by incubation with MysIntα2 antibodies. Suppression of MysIntα2 expression in M. separata larvae by injecting double-stranded RNA also resulted in a decrease in encapsulation activity. Collectively, these results indicate that MysIntα2 plays pivotal roles in the cellular immune response of M. separata, particularly during encapsulation. This likely occurs through the regulation of hemocyte spreading activity, thereby facilitating the formation of multilayered capsules around large invaders.

Modulating macrophage-mediated programmed cell removal: An attractive strategy for cancer therapy.

Macrophage-mediated programmed cell removal (PrCR) is crucial for the identification and elimination of needless cells that maintain tissue homeostasis. The efficacy of PrCR depends on the balance between pro-phagocytic "eat me" signals and anti-phagocytic "don't eat me" signals. Recently, a growing number of studies have shown that tumourigenesis and progression are closely associated with PrCR. In the tumour microenvironment, PrCR activated by the "eat me" signal is counterbalanced by the "don't eat me" signal of CD47/SIRPα, resulting in tumour immune escape. Therefore, targeting exciting "eat me" signalling while simultaneously suppressing "don't eat me" signalling and eventually inducing macrophages to produce effective PrCR will be a very attractive antitumour strategy. Here, we comprehensively review the functions of PrCR-activating signal molecules (CRT, PS, Annexin1, SLAMF7) and PrCR-inhibiting signal molecules (CD47/SIRPα, MHC-I/LILRB1, CD24/Siglec-10, SLAMF3, SLAMF4, PD-1/PD-L1, CD31, GD2, VCAM1), the interactions between these molecules, and Warburg effect. In addition, we highlight the molecular regulatory mechanisms that affect immune system function by exciting or suppressing PrCR. Finally, we review the research advances in tumour therapy by activating PrCR and discuss the challenges and potential solutions to smooth the way for tumour treatment strategies that target PrCR.

Downregulation of Fidgetin-Like 2 Increases Microglial Function: The Relationship Between Microtubules, Morphology, and Activity.

The microtubule cytoskeleton regulates microglial morphology, motility, and effector functions. The microtubule-severing enzyme, fidgetin-like 2 (FL2), negatively regulates cell motility and nerve regeneration, making it a promising therapeutic target for central nervous system injury. Microglia perform important functions in response to inflammation and injury, but how FL2 affects microglia is unclear. In this study, we investigated the role of FL2 in microglial morphology and injury responses in vitro. We first determined that the pro-inflammatory stimulus, lipopolysaccharide (LPS), induced a dose- and time-dependent reduction in FL2 expression associated with reduced microglial ramification. We then administered nanoparticle-encapuslated FL2 siRNA to knockdown FL2 and assess microglial functions compared to negative control siRNA and vehicle controls. Time-lapse live-cell microscopy showed that FL2 knockdown increased the velocity of microglial motility. After incubation with fluorescently labeled IgG-opsonized beads, FL2 knockdown increased phagocytosis. Microglia were exposed to low-dose LPS after nanoparticle treatment to model injury-induced cytokine secretion. FL2 knockdown enhanced LPS-induced cytokine secretion of IL-1α, IL-1ß, and TNFα. These results identify FL2 as a regulator of microglial morphology and suggest that FL2 can be targeted to increase or accelerate microglial injury responses.

Prior Fc receptor activation primes macrophages for increased sensitivity to IgG via long-term and short-term mechanisms.

Macrophages measure the "eat-me" signal immunoglobulin G (IgG) to identify targets for phagocytosis. We tested whether prior encounters with IgG influence macrophage appetite. IgG is recognized by the Fc receptor. To temporally control Fc receptor activation, we engineered an Fc receptor that is activated by the light-induced oligomerization of Cry2, triggering phagocytosis. Using this tool, we demonstrate that subthreshold Fc receptor activation primes mouse bone-marrow-derived macrophages to be more sensitive to IgG in future encounters. Macrophages that have previously experienced subthreshold Fc receptor activation eat more IgG-bound human cancer cells. Increased phagocytosis occurs by two discrete mechanisms-a short- and long-term priming. Long-term priming requires new protein synthesis and Erk activity. Short-term priming does not require new protein synthesis and correlates with an increase in Fc receptor mobility. Our work demonstrates that IgG primes macrophages for increased phagocytosis, suggesting that therapeutic antibodies may become more effective after initial priming doses.