Homeostatic IL-13 in healthy skin directs dendritic cell differentiation to promote TH2 and inhibit TH17 cell polarization.

The signals driving the adaptation of type 2 dendritic cells (DC2s) to diverse peripheral environments remain mostly undefined. We show that differentiation of CD11blo migratory DC2s-a DC2 population unique to the dermis-required IL-13 signaling dependent on the transcription factors STAT6 and KLF4, whereas DC2s in lung and small intestine were STAT6-independent. Similarly, human DC2s in skin expressed an IL-4 and IL-13 gene signature that was not found in blood, spleen and lung DCs. In mice, IL-13 was secreted homeostatically by dermal innate lymphoid cells and was independent of microbiota, TSLP or IL-33. In the absence of IL-13 signaling, dermal DC2s were stable in number but remained CD11bhi and showed defective activation in response to allergens, with diminished ability to support the development of IL-4+GATA3+ helper T cells (TH), whereas antifungal IL-17+RORγt+ TH cells were increased. Therefore, homeostatic IL-13 fosters a noninflammatory skin environment that supports allergic sensitization.

Constant replenishment from circulating monocytes maintains the macrophage pool in the intestine of adult mice.

The paradigm that macrophages that reside in steady-state tissues are derived from embryonic precursors has never been investigated in the intestine, which contains the largest pool of macrophages. Using fate-mapping models and monocytopenic mice, together with bone marrow chimera and parabiotic models, we found that embryonic precursor cells seeded the intestinal mucosa and demonstrated extensive in situ proliferation during the neonatal period. However, these cells did not persist in the intestine of adult mice. Instead, they were replaced around the time of weaning by the chemokine receptor CCR2-dependent influx of Ly6C(hi) monocytes that differentiated locally into mature, anti-inflammatory macrophages. This process was driven largely by the microbiota and had to be continued throughout adult life to maintain a normal intestinal macrophage pool.

Deletion of Tmem268 in mice suppresses anti-infectious immune responses by downregulating CD11b signaling.

Transmembrane protein 268 (TMEM268) is a novel, tumor growth-related protein first reported by our laboratory. It interacts with the integrin subunit ß4 (ITGB4) and plays a positive role in the regulation of the ITGB4/PLEC signaling pathway. Here, we investigated the effects and mechanism of TMEM268 in anti-infectious immune response in mice. Tmem268 knockout in mice aggravated cecal ligation and puncture-induced sepsis, as evidenced by higher bacterial burden in various tissues and organs, congestion, and apoptosis. Moreover, Tmem268 deficiency in mice inhibited phagocyte adhesion and migration, thus decreasing phagocyte infiltration at the site of infection and complement-dependent phagocytosis. Further findings indicated that TMEM268 interacts with CD11b and inhibits its degradation via the endosome-lysosome pathway. Our results reveal a positive regulatory role of TMEM268 in ß2 integrin-associated anti-infectious immune responses and signify the potential value of targeting the TMEM268-CD11b signaling axis for the maintenance of immune homeostasis and immunotherapy for sepsis and related immune disorders.

TALEN-mediated intron editing of HSPCs enables transgene expression restricted to the myeloid lineage.

Gene therapy in hematopoietic stem and progenitor cells (HSPCs) shows great potential for the treatment of inborn metabolic diseases. Typical HSPC gene therapy approaches rely on constitutive promoters to express a therapeutic transgene, which is associated with multiple disadvantages. Here, we propose a novel promoterless intronic gene editing approach that triggers transgene expression only after cellular differentiation into the myeloid lineage. We integrated a splicing-competent eGFP cassette into the first intron of CD11b and observed expression of eGFP in the myeloid lineage but minimal to no expression in HSPCs or differentiated non-myeloid lineages. In vivo, edited HSPCs successfully engrafted in immunodeficient mice and displayed transgene expression in the myeloid compartment of multiple tissues. Using the same approach, we expressed alpha-L-iduronidase (IDUA), the defective enzyme in Mucopolysaccharidosis type I, and observed a 10-fold supraendogenous IDUA expression exclusively after myeloid differentiation. Edited cells efficiently populated bone marrow, blood, and spleen of immunodeficient mice, and retained the capacity to secrete IDUA ex vivo. Importantly, cells edited with the eGFP and IDUA transgenes were also found in the brain. This approach may unlock new therapeutic strategies for inborn metabolic and neurological diseases that require the delivery of therapeutics in brain.

Mechanisms underlying neutrophils adhesion to triple-negative breast cancer cells via CD11b-ICAM1 in promoting breast cancer progression.

BACKGROUND: Triple-negative breast cancer (TNBC) is recognized as the most aggressive and immunologically infiltrated subtype of breast cancer. A high circulating neutrophil-to-lymphocyte ratio (NLR) is strongly linked to a poor prognosis among patients with breast cancer, emphasizing the critical role of neutrophils. Although the involvement of neutrophils in tumor metastasis is well documented, their interactions with primary tumors and tumor cells are not yet fully understood. METHODS: Clinical data were analyzed to investigate the role of neutrophils in breast cancer. In vivo mouse model and in vitro co-culture system were used for mechanism researches. Blocking experiments were further performed to identify therapeutic agents against TNBC. RESULTS: TNBC cells secreted GM-CSF to sustain the survival of mature neutrophils and upregulated CD11b expression. Through CD11b, neutrophils specifically binded to ICAM1 on TNBC cells, facilitating adhesion. Transcriptomic sequencing combined with human and murine functional experiments revealed that neutrophils, through direct CD11b-ICAM1 interactions, activated the MAPK signaling pathway in TNBC cells, thereby enhancing tumor cell invasion and migration. Atorvastatin effectively inhibited ICAM1 expression in tumor cells, and tumor cells with ICAM1 knockout or treated with atorvastatin were unresponsive to neutrophil activation. The MAPK pathway and MMP9 expression were significantly inhibited in the tumor tissues of TNBC patients treated with atorvastatin. CONCLUSIONS: Targeting CD11b-ICAM1 with atorvastatin represented a potential clinical approach to reduce the malignant characteristics of TNBC.

Daytime Light Deficiency Leads to Sex- and Brain Region-Specific Neuroinflammatory Responses in a Diurnal Rodent.

Seasonal changes in peripheral inflammation are well documented in both humans and animal models, but seasonal changes in neuroinflammation, especially the impact of seasonal lighting environment on neuroinflammation remain unclear. To address this question, the present study examined the effects of environmental lighting conditions on neuroinflammation in a diurnal rodent model, Nile grass rats (Arvicanthis niloticus). Male and female grass rats were housed in either bright (brLD) or dim (dimLD) light during the day to simulate a summer or winter light condition, respectively. After 4 weeks, microglia markers Iba-1 and CD11b, as well as pro-inflammatory cytokines TNF-α and IL-6, were examined in the anterior cingulate cortex (ACC), basolateral amygdala (BLA), and dorsal hippocampus (dHipp). The results revealed that winter-like dim light during the day leads to indicators of increased neuroinflammation in a brain site- and sex-specific manner. Specifically, relatively few changes in the neuroinflammatory markers were observed in the ACC, while numerous changes were found in the BLA and dHipp. In the BLA, winter-like dimLD resulted in hyper-ramified microglia morphology and increased expression of the pro-inflammatory cytokine IL-6, but only in males. In the dHipp, dimLD led to a higher number and hyper-ramified morphology of microglia as well as increased expression of CD11b and TNF-α, but only in females. Neuroinflammatory state is thus influenced by environmental light, differently in males and females, and could play a role in sex differences in the prevalence and symptoms of psychiatric or neurological disorders that are influenced by season or other environmental light conditions. Diurnal Nile grass rats were housed under bright or dim light during the day for 4 weeks, simulating seasonal fluctuations in daytime lighting environment. Dim light housing resulted in hyper-ramified morphology of microglia (scale bar, 15 µm) and altered expression of pro-inflammatory cytokines (TNF-α) in a sex- and brain region-specific manner.

Integrin CD11b Negatively Regulates B Cell Receptor Signaling to Shape Humoral Response during Immunization and Autoimmunity.

Our previous work has revealed the ability of CD11b to regulate BCR signaling and control autoimmune disease in mice. However, how CD11b regulates the immune response under normal conditions remains unknown. Through the use of a CD11b knockout model on a nonautoimmune background, we demonstrated that CD11b-deficient mice have an elevated Ag-specific humoral response on immunization. Deletion of CD11b resulted in elevated low-affinity and high-affinity IgG Ab and increases in Ag-specific germinal center B cells and plasma cells (PCs). Examination of BCR signaling in CD11b-deficient mice revealed defects in association of negative regulators pLyn and CD22 with the BCR, but increases in colocalizations between positive regulator pSyk and BCR after stimulation. Using a CD11b-reporter mouse model, we identified multiple novel CD11b-expressing B cell subsets that are dynamically altered during immunization. Subsequent experiments using a cell-specific CD11b deletion model revealed this effect to be B cell intrinsic and not altered by myeloid cell CD11b expression. Importantly, CD11b expression on PCs also impacts on BCR repertoire selection and diversity in autoimmunity. These studies describe a novel role for CD11b in regulation of the healthy humoral response and autoimmunity, and reveal previously unknown populations of CD11b-expressing B cell subsets, suggesting a complex function for CD11b in B cells during development and activation.

CD11b Deficiency Favors Cartilage Calcification via Increased Matrix Vesicles, Apoptosis, and Lysyl Oxidase Activity.

Pathological cartilage calcification is a hallmark feature of osteoarthritis, a common degenerative joint disease, characterized by cartilage damage, progressively causing pain and loss of movement. The integrin subunit CD11b was shown to play a protective role against cartilage calcification in a mouse model of surgery-induced OA. Here, we investigated the possible mechanism by which CD11b deficiency could favor cartilage calcification by using naïve mice. First, we found by transmission electron microscopy (TEM) that CD11b KO cartilage from young mice presented early calcification spots compared with WT. CD11b KO cartilage from old mice showed progression of calcification areas. Mechanistically, we found more calcification-competent matrix vesicles and more apoptosis in both cartilage and chondrocytes isolated from CD11b-deficient mice. Additionally, the extracellular matrix from cartilage lacking the integrin was dysregulated with increased collagen fibrils with smaller diameters. Moreover, we revealed by TEM that CD11b KO cartilage had increased expression of lysyl oxidase (LOX), the enzyme that catalyzes matrix crosslinks. We confirmed this in murine primary CD11b KO chondrocytes, where Lox gene expression and crosslinking activity were increased. Overall, our results suggest that CD11b integrin regulates cartilage calcification through reduced MV release, apoptosis, LOX activity, and matrix crosslinking. As such, CD11b activation might be a key pathway for maintaining cartilage integrity.

A novel ZsGreen knock-in melanoma cell line reveals the function of CD11b in tumor phagocytosis.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) is an efficient tool for establishing genetic models including cellular models, and has facilitated unprecedented advancements in biomedical research. In both patients and cancer animal models, immune cells infiltrate the tumor microenvironment and some of them migrate to draining lymph nodes to exert antitumor effects. Among these immune cells, phagocytes such as macrophages and dendritic cells engulf tumor antigens prior to their crosstalk with T cells and elicit adaptive immune response against tumors. Melanoma cells are frequently used as a tumor model because of their relatively high level of somatic mutations and antigenicity. However, few genetic models have been developed using melanoma cell lines to track tumor cell phagocytosis, which is essential for understanding protective immune response in vivo. In this study, we used CRISPR/Cas9-mediated DNA cleavage and homologous recombination to develop a novel knock-in tool which expresses the ultra-bright fluorescent probe ZsGreen in YUMM1.7 melanoma cells. Using this novel tool, we measured the macrophagic engulfment of melanoma cells inside the tumor microenvironment. We also found that in tumor-grafted mice, a subset of dendritic cells efficiently engulfed YUMM1.7 cells and was preferentially trafficking tumor antigens to draining lymph nodes. In addition, we used this knock-in tool to assess the impact of a point mutation of CD11b on phagocytosis in the tumor microenvironment. Our results demonstrate that the ZsGreen-expressing YUMM1.7 melanoma model provides a valuable tool for the study of phagocytosis in vivo.

Conventional and monocyte-derived CD11b(+) dendritic cells initiate and maintain T helper 2 cell-mediated immunity to house dust mite allergen.

Dendritic cells (DCs) are crucial for mounting allergic airway inflammation, but it is unclear which subset of DCs performs this task. By using CD64 and MAR-1 staining, we reliably separated CD11b(+) monocyte-derived DCs (moDCs) from conventional DCs (cDCs) and studied antigen uptake, migration, and presentation assays of lung and lymph node (LN) DCs in response to inhaled house dust mite (HDM). Mainly CD11b(+) cDCs but not CD103(+) cDCs induced T helper 2 (Th2) cell immunity in HDM-specific T cells in vitro and asthma in vivo. Studies in Flt3l(-/-) mice, lacking all cDCs, revealed that moDCs were also sufficient to induce Th2 cell-mediated immunity but only when high-dose HDM was given. The main function of moDCs was the production of proinflammatory chemokines and allergen presentation in the lung during challenge. Thus, we have identified migratory CD11b(+) cDCs as the principal subset inducing Th2 cell-mediated immunity in the LN, whereas moDCs orchestrate allergic inflammation in the lung.

A Genetic Model of Constitutively Active Integrin CD11b/CD18.

Pharmacological activation of integrin CD11b/CD18 (αMß2, Mac-1, and CR3) shows anti-inflammatory benefits in a variety of animal models of human disease, and it is a novel therapeutic strategy. Reasoning that genetic models can provide an orthogonal and direct system for the mechanistic study of CD11b agonism, we present in this study, to our knowledge, a novel knock-in model of constitutive active CD11b in mice. We genetically targeted the Itgam gene (which codes for CD11b) to introduce a point mutation that results in the I332G substitution in the protein. The I332G mutation in CD11b promotes an active, higher-affinity conformation of the ligand-binding I/A-domain (CD11b αA-domain). In vitro, this mutation increased adhesion of knock-in neutrophils to fibrinogen and decreased neutrophil chemotaxis to a formyl-Met-Leu-Phe gradient. In vivo, CD11bI332G animals showed a reduction in recruitment of neutrophils and macrophages in a model of sterile peritonitis. This genetic activation of CD11b also protected against development of atherosclerosis in the setting of hyperlipidemia via reduction of macrophage recruitment into atherosclerotic lesions. Thus, our animal model of constitutive genetic activation of CD11b can be a useful tool for the study of integrin activation and its potential contribution to modulating leukocyte recruitment and alleviating different inflammatory diseases.

Altered Macrophage Function Associated with Crystalline Lung Inflammation in Acid Sphingomyelinase Deficiency.

Deficiency of ASM (acid sphingomyelinase) causes the lysosomal storage Niemann-Pick disease (NPD). Patients with NPD type B may develop progressive interstitial lung disease with frequent respiratory infections. Although several investigations using the ASM-deficient (ASMKO) mouse NPD model revealed inflammation and foamy macrophages, there is little insight into the pathogenesis of NPD-associated lung disease. Using ASMKO mice, we report that ASM deficiency is associated with a complex inflammatory phenotype characterized by marked accumulation of monocyte-derived CD11b+ macrophages and expansion of airspace/alveolar CD11c+ CD11b- macrophages, both with increased size, granularity, and foaminess. Both the alternative and classical pathways were activated, with decreased in situ phagocytosis of opsonized (Fc-coated) targets, preserved clearance of apoptotic cells (efferocytosis), secretion of Th2 cytokines, increased CD11c+/CD11b+ cells, and more than a twofold increase in lung and plasma proinflammatory cytokines. Macrophages, neutrophils, eosinophils, and noninflammatory lung cells of ASMKO lungs also exhibited marked accumulation of chitinase-like protein Ym1/2, which formed large eosinophilic polygonal Charcot-Leyden-like crystals. In addition to providing insight into novel features of lung inflammation that may be associated with NPD, our report provides a novel connection between ASM and the development of crystal-associated lung inflammation with alterations in macrophage biology.

Trajectory of change in brain complement factors from neonatal to young adult humans.

Immune system components also regulate synapse formation and refinement in neurodevelopment. The complement pathway, associated with cell lysis and phagocytosis, is implicated in synaptic elimination. Aberrant adolescent synaptic pruning may underpin schizophrenia onset; thus, changes in cortical complement activity during human development are of major interest. Complement is genetically linked to schizophrenia via increased C4 copy number variants, but the developmental trajectory of complement expression in the human brain is undetermined. As complement increases during periods of active synaptic engulfment in rodents, we hypothesized that complement expression would increase during postnatal development in humans, particularly during adolescence. Using human postmortem prefrontal cortex, we observed that complement activator (C1QB and C3) transcripts peaked in early neurodevelopment, and were highest in toddlers, declining in teenagers (all ANCOVAs between F = 2.41 -3.325, p = .01-0.05). We found that C4 protein was higher at 1-5 years (H = 16.378, p = .012), whereas C3 protein levels were unchanged with age. The microglial complement receptor subunit CD11b increased in mRNA early in life and peaked in the toddler brain (ANCOVA: pH, F = 4.186, p = .003). Complement inhibitors (CD46 and CD55) increased at school age, but failed to decrease like complement activators (both ANCOVAs, F > 4.4, p < .01). These data suggest the activation of complement in the human prefrontal cortex occurs between 1 and 5 years. We did not find evidence of induction of complement factors during adolescence and instead found increased or sustained levels of complement inhibitor mRNA at maturation. Dysregulation of these typical patterns of complement may predispose the brain to neurodevelopmental disorders such as autism or schizophrenia.

Placental multipotent mesenchymal stromal cell-derived Slit2 may regulate macrophage motility during placental infection.

Slit proteins have been reported to act as axonal repellents in Drosophila; however, their role in the placental microenvironment has not been explored. In this study, we found that human placental multipotent mesenchymal stromal cells (hPMSCs) constitutively express Slit2. Therefore, we hypothesized that Slit2 expressed by hPMSCs could be involved in macrophage migration during placental inflammation through membrane cognate Roundabout (Robo) receptor signaling. In order to develop a preclinical in vitro mouse model of hPMSCs in treatment of perinatal infection, RAW 264.7 cells were used in this study. Slit2 interacted with Robo4 that was highly expressed in RAW 264.7 macrophages: their interaction increased the adhesive ability of RAW 264.7 cells and inhibited migration. Lipopolysaccharide (LPS)-induced CD11bCD18 expression could be inhibited by Slit2 and by hPMSC-conditioned medium (CM). LPS-induced activation of p38 and Rap1 was also attenuated by Slit2 and by hPMSC-CM. Noticeably, these inhibitory effects of hPMSC-CM decreased after depletion of Slit2 from the CM. Furthermore, we found that p38 siRNA inhibited LPS-induced Rap1 expression in RAW 264.7 cells, indicating that Rap1 functions downstream of p38 signaling. p38 siRNA increased cell adhesion and inhibited migration through reducing LPS-stimulated CD11bCD18 expression in RAW 264.7 cells. Thus, hPMSC-derived Slit2 may inhibit LPS-induced CD11bCD18 expression to decrease cell migration and increase adhesion through modulating the activity and motility of inflammatory macrophages in placenta. This may represent a novel mechanism for LPS-induced placental infection.

Estrogens and the Angiotensin II Type 2 Receptor Control Flow-Mediated Outward Remodeling in the Female Mouse Mesenteric Artery.

Flow-mediated outward remodeling (FMR) is involved in postischemic revascularization. Angiotensin II type 2 receptor (AT2R), through activation of T-cell-mediated IL-17 production, and estrogens are involved in FMR. Thus, we investigated the interplay between estrogens and AT2R in FMR using a model of ligation of feed arteries supplying collateral pathways in mouse mesenteric arteries in vivo. Arteries were collected after 2 (inflammatory phase), 4 (diameter expansion phase), and 7 days (remodeling completed). We used AT2R+/+ and AT2R-/- ovariectomized (OVX) female mice treated or not with 17-beta-estradiol (E2). Seven days after ligation, arterial diameter was larger in high flow (HF) compared to normal flow (NF) arteries. FMR was absent in OVX mice and restored by E2. AT2R gene expression was higher in HF than in NF arteries only in E2-treated OVX AT2R+/+ mice. CD11b and TNF alpha levels (inflammatory phase), MMP2 and TIMP1 (extracellular matrix digestion), and NOS3 (diameter expansion phase) expression levels were higher in HF than in NF arteries only in E2-treated AT2R+/+ mice, not in the other groups. Thus, E2 is necessary for AT2R-dependent diameter expansion, possibly through activation of T-cell AT2R, in arteries submitted chronically to high blood flow.

Differentiation of c-Kit+ CD24+ natural killer cells into myeloid cells in a GATA-2-dependent manner.

Myeloid progenitor cells have generally been considered the predominant source of myeloid cells under steady-state conditions. Here we show that NK cells contributed to a myeloid cell lineage pool in naïve and tumor-bearing mice. Using fate tracing of NKp46+ cells, we found that myeloid cells could be derived from NK cells. Notably, among mature CD11b+ CD27+ NK cells, c-Kit+ CD24+ NK cells were capable of differentiating into a range of myeloid lineages in vitro and produced neutrophils and monocytes in vivo. The differentiation was completely inhibited by NK-stimulating cytokines. In addition to the potential for differentiation into myeloid cells, c-Kit+ CD24+ NK cells retained NK cell phenotypes and effector functions. Mechanistically, GATA-2 was necessary for the differentiation of c-Kit+ CD24+ NK cells. Therefore, we discovered that GATA-2-dependent differentiation of c-Kit+ CD24+ NK cells contributes to myeloid cell development and identified a novel pathway for myeloid lineage commitment under physiological conditions.

Dysfunction of complement receptors CR3 (CD11b/18) and CR4 (CD11c/18) in pre-eclampsia: a genetic and functional study.

OBJECTIVE: To study genetic variants and their function within genes coding for complement receptors in pre-eclampsia. DESIGN: A case-control study. SETTING: Pre-eclampsia is a common vascular disease of pregnancy. The clearance of placenta-derived material is one of the functions of the complement system in pregnancy. POPULATION: We genotyped 500 women with pre-eclamptic pregnancies and 190 pregnant women without pre-eclampsia, as controls, from the FINNPEC cohort, and 122 women with pre-eclamptic pregnancies and 1905 controls from the national FINRISK cohort. METHODS: The functional consequences of genotypes discovered by targeted exomic sequencing were explored by analysing the binding of the main ligand iC3b to mutated CR3 or CR4, which were transiently expressed on the surface of COS-1 cells. MAIN OUTCOME MEASURES: Allele frequencies were compared between pre-eclamptic pregnancies and controls in genetic studies. The functional consequences of selected variants were measured by binding assays. RESULTS: The most significantly pre-eclampsia-linked CR3 variant M441K (P = 4.27E-4, OR = 1.401, 95% CI = 1.167-1.682) displayed a trend of increased adhesion to iC3b (P = 0.051). The CR4 variant A251T was found to enhance the adhesion of CR4 to iC3b, whereas W48R resulted in a decrease of the binding of CR4 to iC3b. CONCLUSIONS: Results suggest that changes in complement-facilitated phagocytosis are associated with pre-eclampsia. Further studies are needed to ascertain whether aberrant CR3 and CR4 activity leads to altered pro- and anti-inflammatory cytokine responses in individuals carrying the associated variants, and the role of these receptors in pre-eclampsia pathogenesis. TWEETABLE ABSTRACT: Genetic variants of complement receptors CR3 and CR4 have functional consequences that are associated with pre-eclampsia.

Annexin A3 as a Marker Protein for Microglia in the Central Nervous System of Rats.

The parenchymal microglia possess different morphological characteristics in cerebral physiological and pathological conditions; thus, visualizing these cells is useful as a means of further investigating parenchymal microglial function. Annexin A3 (ANXA3) is expressed in microglia, but it is unknown whether it can be used as a marker protein for microglia and its physiological function. Here, we compared the distribution and morphology of parenchymal microglia labeled by ANXA3, cluster of differentiation 11b (CD11b), and ionized calcium-binding adaptor molecule 1 (Iba1) and measured the expression of ANXA3 in nonparenchymal macrophages (meningeal and perivascular macrophages). We also investigated the spatiotemporal expression of ANXA3, CD11b, and Iba1 in vivo and in vitro and the cellular function of ANXA3 in microglia. We demonstrated that ANXA3-positive cells were abundant and evenly distributed throughout the whole brain tissue and spinal cord of adult rats. The morphology and distribution of ANXA3-labeled microglia were quite similar to those labeled by the microglial-specific markers CD11b and Iba1 in the central nervous system (CNS). ANXA3 was expressed in the cytoplasm of microglia, and its expression was significantly increased in activated microglia. ANXA3 was almost undetectable in the nonparenchymal macrophages. Meanwhile, the protein and mRNA expression levels of ANXA3 in different regions of the CNS were different from those of CD11b and Iba1. Moreover, knockdown of ANXA3 inhibited the proliferation and migration of microglia, while overexpression of ANXA3 enhanced these activities. This study confirms that ANXA3 may be a novel marker for parenchymal microglia in the CNS of adult rats and enriches our understanding of ANXA3 from expression patterns to physiological function.

Complement-Opsonized Nano-Carriers Are Bound by Dendritic Cells (DC) via Complement Receptor (CR)3, and by B Cell Subpopulations via CR-1/2, and Affect the Activation of DC and B-1 Cells.

The development of nanocarriers (NC) for biomedical applications has gained large interest due to their potential to co-deliver drugs in a cell-type-targeting manner. However, depending on their surface characteristics, NC accumulate serum factors, termed protein corona, which may affect their cellular binding. We have previously shown that NC coated with carbohydrates to enable biocompatibility triggered the lectin-dependent complement pathway, resulting in enhanced binding to B cells via complement receptor (CR)1/2. Here we show that such NC also engaged all types of splenic leukocytes known to express CR3 at a high rate when NC were pre-incubated with native mouse serum resulting in complement opsonization. By focusing on dendritic cells (DC) as an important antigen-presenting cell type, we show that CR3 was essential for binding/uptake of complement-opsonized NC, whereas CR4, which in mouse is specifically expressed by DC, played no role. Further, a minor B cell subpopulation (B-1), which is important for first-line pathogen responses, and co-expressed CR1/2 and CR3, in general, engaged NC to a much higher extent than normal B cells. Here, we identified CR-1/2 as necessary for binding of complement-opsonized NC, whereas CR3 was dispensable. Interestingly, the binding of complement-opsonized NC to both DC and B-1 cells affected the expression of activation markers. Our findings may have important implications for the design of nano-vaccines against infectious diseases, which codeliver pathogen-specific protein antigen and adjuvant, aimed to induce a broad adaptive cellular and humoral immune response by inducing cytotoxic T lymphocytes that kill infected cells and pathogen-neutralizing antibodies, respectively. Decoration of nano-vaccines either with carbohydrates to trigger complement activation in vivo or with active complement may result in concomitant targeting of DC and B cells and thereby may strongly enhance the extent of dual cellular/humoral immune responses.

Microglia Promote Increased Pain Behavior through Enhanced Inflammation in the Spinal Cord during Repeated Social Defeat Stress.

Clinical studies indicate that psychosocial stress contributes to adverse chronic pain outcomes in patients, but it is unclear how this is initiated or amplified by stress. Repeated social defeat (RSD) is a mouse model of psychosocial stress that activates microglia, increases neuroinflammatory signaling, and augments pain and anxiety-like behaviors. We hypothesized that activated microglia within the spinal cord facilitate increased pain sensitivity following RSD. Here we show that mechanical allodynia in male mice was increased with exposure to RSD. This stress-induced behavior corresponded with increased mRNA expression of several inflammatory genes, including IL-1ß, TNF-α, CCL2, and TLR4 in the lumbar spinal cord. While there were several adhesion and chemokine-related genes increased in the lumbar spinal cord after RSD, there was no accumulation of monocytes or neutrophils. Notably, there was evidence of microglial activation selectively within the nociceptive neurocircuitry of the dorsal horn of the lumbar cord. Elimination of microglia using the colony stimulating factor 1 receptor antagonist PLX5622 from the brain and spinal cord prevented the development of mechanical allodynia in RSD-exposed mice. Microglial elimination also attenuated RSD-induced IL-1ß, CCR2, and TLR4 mRNA expression in the lumbar spinal cord. Together, RSD-induced allodynia was associated with microglia-mediated inflammation within the dorsal horn of the lumbar spinal cord.SIGNIFICANCE STATEMENT Mounting evidence indicates that psychological stress contributes to the onset and progression of adverse nociceptive conditions. We show here that repeated social defeat stress causes increased pain sensitivity due to inflammatory signaling within the nociceptive circuits of the spinal cord. Studies here mechanistically tested the role of microglia in the development of pain by stress. Pharmacological ablation of microglia prevented stress-induced pain sensitivity. These findings demonstrate that microglia are critical mediators in the induction of pain conditions by stress. Moreover, these studies provide a proof of principle that microglia can be targeted as a therapeutic strategy to mitigate adverse pain conditions.