CX3CR1 Deficiency Research Articles

Pravastatin prevents colitis-associated carcinogenesis by reducing CX3CR1high M2-like fibrocyte counts in the inflamed colon

Colorectal cancer (CRC) resulting from chronic inflammation is a crucial issue in patients with inflammatory bowel disease (IBD). Although many reports established that intestinal resident CX3CR1high macrophages play an essential role in suppressing intestinal inflammation, their function in colitis-related CRC remains unclear. In this study, we found that colonic CX3CR1high macrophages, which were positive for MHC-II, F4/80 and CD319, promoted colitis-associated CRC. They highly expressed Col1a1, Tgfb, II10, and II4, and were considered to be fibrocytes with an immunosuppressive M2-like phenotype. CX3CR1 deficiency led to reductions in the absolute numbers of CX3CR1high fibrocytes through increased apoptosis, thereby preventing the development of colitis-associated CRC. We next focused statins as drugs targeting CX3CR1high fibrocytes. Statins have been actively discussed for patients with IBD and reported to suppress the CX3CL1/CX3CR1 axis. Statin treatment after azoxymethane/dextran sulfate sodium-induced inflammation reduced CX3CR1high fibrocyte counts and suppressed colitis-associated CRC. Therefore, CX3CR1high fibrocytes represent a potential target for carcinogenesis-preventing therapy, and statins could be safe therapeutic candidates for IBD.

Detrimental Role of CXCR3 in α-Naphthylisothiocyanate- and Triptolide-Induced Cholestatic Liver Injury.

The chemokine receptor CXCR3 is functionally pleiotropic, not only recruiting immune cells to the inflamed liver but also mediating the pathological process of cholestatic liver injury (CLI). However, the mechanism of its involvement in the CLI remains unclear. Both alpha-naphthylisothiocyanate (ANIT) and triptolide are hepatotoxicants that induce CLI by bile acid (BA) dysregulation, inflammation, and endoplasmic reticulum (ER)/oxidative stress. Through molecular docking, CXCR3 is a potential target of ANIT and triptolide. Therefore, this study aimed to investigate the role of CXCR3 in ANIT- and triptolide-induced CLI and to explore the underlying mechanisms. Wild-type mice and CXCR3-deficient mice were administered with ANIT or triptolide to compare CLI, BA profile, hepatic recruitment of IFN-γ/IL-4/IL-17+CD4+T cells, IFN-γ/IL-4/IL-17+iNKT cells and IFN-γ/IL-4+NK cells, and the expression of ER/oxidative stress pathway. The results showed that CXCR3 deficiency ameliorated ANIT- and triptolide-induced CLI. CXCR3 deficiency alleviated ANIT-induced dysregulated BA metabolism, which decreased the recruitment of IFN-γ+NK cells and IL-4+NK cells to the liver and inhibited ER stress. After triptolide administration, CXCR3 deficiency ameliorated dysregulation of BA metabolism, which reduced the migration of IL-4+iNKT cells and IL-17+iNKT cells and reduced oxidative stress through inhibition of Egr1 expression and AKT phosphorylation. Our findings suggest a detrimental role of CXCR3 in ANIT- and triptolide-induced CLI, providing a promising therapeutic target and introducing novel mechanisms for understanding cholestatic liver diseases.

Cx3cr1 deficiency interferes with learning- and direct current stimulation-mediated neuroplasticity of the motor cortex.

Microglia are essential contributors to synaptic transmission and stability and communicate with neurons via the fractalkine pathway. Transcranial direct current stimulation [(t)DCS], a form of non-invasive electrical brain stimulation, modulates cortical excitability and promotes neuroplasticity, which has been extensively demonstrated in the motor cortex and for motor learning. The role of microglia and their fractalkine receptor CX3CR1 in motor cortical neuroplasticity mediated by DCS or motor learning requires further elucidation. We demonstrate the effects of pharmacological microglial depletion and genetic Cx3cr1 deficiency on the induction of DCS-induced long-term potentiation (DCS-LTP) ex vivo. The relevance of microglia-neuron communication for DCS response and structural neuroplasticity underlying motor learning are assessed via 2-photon in vivo imaging. The behavioural consequences of impaired CX3CR1 signalling are investigated for both gross and fine motor learning. We show that DCS-mediated neuroplasticity in the motor cortex depends on the presence of microglia and is driven in part by CX3CR1 signalling ex vivo and provide the first evidence of microglia interacting with neurons during DCS in vivo. Furthermore, CX3CR1 signalling is required for motor learning and underlying structural neuroplasticity in concert with microglia interaction. Although we have recently demonstrated the microglial response to DCS in vivo, we now provide a link between microglial integrity and neuronal activity for the expression of DCS-dependent neuroplasticity. In addition, we extend the knowledge on the relevance of CX3CR1 signalling for motor learning and structural neuroplasticity. The underlying molecular mechanisms and the potential impact of DCS in rescuing CX3CR1 deficits remain to be addressed in the future.

CXCR3 deficiency decreases autoantibody production by inhibiting aberrant activated T follicular helper cells and B cells in lupus mice

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a high level of autoantibody production. T follicular helper (Tfh) cells and B cells participate in the development of SLE. Several studies have shown that CXCR3+ cells are increased in SLE patients. However, the mechanism through which CXCR3 influences lupus development remains unclear. In this study, we established lupus models to determine the role of CXCR3 in lupus pathogenesis. The concentration of autoantibodies was detected using the enzyme-linked immunosorbent assay (ELISA), and the percentages of Tfh cells and B cells were measured using flow cytometry. RNA sequencing (RNA-seq) was performed to detect the differentially expressed genes in CD4+ T cells from wild-type (WT) and CXCR3 knock-out (KO) lupus mice. Migration of CD4+ T cells in spleen section was assessed using immunofluorescence. CD4+ T cell function in helping B cells produce antibodies was determined using a co-culture experiment and supernatant IgG ELISA. Lupus mice were treated with a CXCR3 antagonist to confirm the therapeutic effects. We found that the expression of CXCR3 was increased in CD4+ T cells from lupus mice. CXCR3 deficiency reduced autoantibody production with decreased proportions of Tfh cells, germinal center (GC) B cells, and plasma cells. Expression of Tfh-related genes was downregulated in CD4+ T cells from CXCR3 KO lupus mice. Migration to B cell follicles and T-helper function of CD4+ T cells were reduced in CXCR3 KO lupus mice. CXCR3 antagonist AMG487 decreased the level of serum anti-dsDNA IgG in lupus mice. We clarify that CXCR3 may play an important role in autoantibody production by increasing the percentages of aberrant activated Tfh cells and B cells and promoting the migration and T-helper function of CD4+ T cells in lupus mice. Thus, CXCR3 may be a potential target for lupus therapy.

5-aminolevulinate and CHIL3/CHI3L1 treatment amid ischemia aids liver metabolism and reduces ischemia-reperfusion injury.

Rationale: Liver resection and transplantation surgeries are accompanied by hepatic ischemia-reperfusion (HIR) injury that hampers the subsequent liver recovery. Given that the liver is the main organ for metabolism and detoxification, ischemia-reperfusion in essence bestows metabolic stress upon the liver and disrupts local metabolic and immune homeostasis. Most of the recent and current research works concerning HIR have been focusing on addressing HIR-induced hepatic injury and inflammation, instead of dealing with the metabolic reprogramming and restoration of redox homeostasis. As our previous work uncovers the importance of 5-aminolevulinate (5-ALA) synthesis during stress adaptation, here we evaluate the effects of supplementing 5-ALA to mitigate HIR injury. Methods: 5-ALA was supplemented into the mice or cultured cells during the ischemic or oxygen-glucose deprivation (OGD) phase. Following reperfusion or reoxygenation, cellular metabolism and energy homeostasis, mitochondrial production of reactive oxygen species (ROS) and transcriptomic changes were evaluated in HIR mouse models or cultured hepatocytes and macrophages. Liver injury, hepatocytic functional tests, and macrophagic M1/M2 polarization were assessed. Results: Dynamic changes in the expression of key enzymes in 5-ALA metabolism were first confirmed in donor and mouse liver samples following HIR. Supplemented 5-ALA modulated mouse hepatic lipid metabolism and reduced ATP production in macrophages following HIR, resulting in elevation of anti-inflammatory M2 polarization. Mechanistically, 5-ALA down-regulates macrophagic chemokine receptor CX3CR1 via the repression of RelA following OGD and reoxygenation (OGD/R). Cx3cr1 KO mice demonstrated milder liver injuries and more macrophage M2 polarization after HIR. M2 macrophage-secreted chitinase-like protein 3 (CHIL3; CHI3L1 in human) is an important HIR-induced effector downstream of CX3CR1 deficiency. Addition of CHIL3/CHI3L1 alone improved hepatocellular metabolism and reduced OGD/R-inflicted injuries in cultured mouse and human hepatocytes. Combined treatment with 5-ALA and CHIL3 during the ischemic phase facilitated lipid metabolism and ATP production in the mouse liver following HIR. Conclusion: Our results reveal that supplementing 5-ALA promotes macrophagic M2 polarization via downregulation of RelA and CX3CR1 in mice following HIR, while M2 macrophage-produced CHIL3/CHI3L1 also manifests beneficial effects to the recovery of hepatic metabolism. 5-ALA and CHIL3/CHI3L1 together mitigate HIR-induced mitochondrial dysfunction and hepatocellular injuries, which may be developed into safe and effective clinical treatments to attenuate HIR injuries.

CX3CR1 deficiency leads to impairment of immune surveillance in the epididymis.

Mononuclear phagocytes (MPs) play an active role in the immunological homeostasis of the urogenital tract. In the epididymis, a finely tuned balance between tolerance to antigenic sperm and immune activation is required to maintain epididymal function while protecting sperm against pathogens and stressors. We previously characterized a subset of resident MPs that express the CX3CR1 receptor, emphasizing their role in antigen sampling and processing during sperm maturation and storage in the murine epididymis. Bacteria-associated epididymitis is the most common cause of intrascrotal inflammation and frequently leads to reproductive complications. Here, we examined whether the lack of functional CX3CR1 in homozygous mice (CX3CR1EGFP/EGFP, KO) alters the ability of MPs to initiate immune responses during epididymitis induced by LPS intravasal-epididymal injection. Confocal microscopy revealed that CX3CR1-deficient MPs located in the initial segments of the epididymis displayed fewer luminal-reaching membrane projections and impaired antigen capture activity. Moreover, flow cytometry showed a reduction of epididymal KO MPs with a monocytic phenotype under physiological conditions. In contrast, flow cytometry revealed an increase in the abundance of MPs with a monocytic signature in the distal epididymal segments after an LPS challenge. This was accompanied by the accumulation of CD103+ cells in the interstitium, and the prevention or attenuation of epithelial damage in the KO epididymis during epididymitis. Additionally, CX3CR1 deletion induced downregulation of Gja1 (connexin 43) expression in KO MPs. Together, our study provides evidence that MPs are gatekeepers of the immunological blood-epididymis barrier and reveal the role of the CX3CR1 receptor in epididymal mucosal homeostasis by inducing MP luminal protrusions and by regulating the monocyte population in the epididymis at steady state as well as upon infection. We also uncover the interaction between MPs and CD103+ dendritic cells, presumably through connexin 43, that enhance immune responses during epididymitis. Our study may lead to new diagnostics and therapies for male infertility and epididymitis by identifying immune mechanisms in the epididymis.

Cx3cr1 deficiency aggravates Aβ driven neurodegeneration and cognitive decline in a mouse model of Alzheimer's disease.

AbstractBackgroundDespite its identification as a key checkpoint regulator of microglial activation in Alzheimer’s disease, the overarching role of CX3CR1 signaling in modulating mechanisms of Aβ driven neurodegeneration, including accumulation of hyperphosphorylated tau in is not well understood.MethodUsing 4 ‐ and 6‐month old 5xFAD;Cx3cr1+/+ and 5xFAD;Cx3cr1‐/‐ mice, we use immunohistochemistry, western blotting, transcriptomic analysis and quantitative real time PCR to investigate accumulation of soluble and insoluble Aβ species, plaque associated microglial activation, synaptic dysregulation and neurodegeneration with disease progression. Flow cytometry based, Aβ uptake assays are used for in‐vivo characterization of the effects of CX3CR1‐signaling on microglial phagocytosis and degradation of methoxy‐X04+ fibrillar Aβ. Lastly, we use Y‐maze testing to analyze the effects of Cx3cr1 deficiency on working memory in 5xFAD mice.ResultWe show that disease progression in 5xFAD;Cx3cr1‐/‐ mice is characterized by increased deposition of filamentous plaques that display defective microglial plaque engagement, along with impaired microglial Aβ phagocytosis and lysosomal acidification in‐vivo. Interestingly, Cx3cr1 deficiency resulted in heighted accumulation of neurotoxic, oligomeric Aβ, which correlated with severe neuritic dystrophy, preferential loss of post‐synaptic densities, exacerbated tau pathology, neuronal loss and cognitive impairment. Transcriptomic analyses using cortical RNA coupled with real time PCR validation using FACS‐purified microglial cells revealed dysregulated TGFβ responses, inflammatory signaling and oxidative stress responses in 5xFAD;Cx3cr1‐/‐ mice.ConclusionIn contrast to increased Aβ uptake by Cx3cr1‐/‐ microglia during early disease, Cx3cr1 deficiency impairs microglial uptake and degradation of fibrillar Aβ, thereby triggering an accumulation of neurotoxic soluble and insoluble Aβ species with disease progression. Our results indicate that Aβ‐driven microglial dysfunction in Cx3cr1‐/‐ mice aggravates tau hyperphosphorylation, neurodegeneration, synaptic dysregulation and impairs working memory. This study underscores the importance of investigating the time‐dependent role of CX3CR1‐driven microglial responses in AD.

CXCR2 intrinsically drives the maturation and function of neutrophils in mice.

Neutrophils play a major role in the protection from infections but also in inflammation related to tumor microenvironment. However, cell-extrinsic and -intrinsic cues driving their function at steady state is still fragmentary. Using Cxcr2 knock-out mice, we have evaluated the function of the chemokine receptor Cxcr2 in neutrophil physiology. We show here that Cxcr2 deficiency decreases the percentage of mature neutrophils in the spleen, but not in the bone marrow (BM). There is also an increase of aged CD62Llo CXCR4hi neutrophils in the spleen of KO animals. Spleen Cxcr2-/- neutrophils display a reduced phagocytic ability, whereas BM neutrophils show an enhanced phagocytic ability compared to WT neutrophils. Spleen Cxcr2-/- neutrophils show reduced reactive oxygen species production, F-actin and α-tubulin levels. Moreover, spleen Cxcr2-/- neutrophils display an altered signaling with reduced phosphorylation of ERK1/2 and p38 MAPK, impaired PI3K-AKT, NF-κB, TGFβ and IFNγ pathways. Altogether, these results suggest that Cxcr2 is essential for neutrophil physiology.

CX3CL1/CX3CR1 interaction protects against lipotoxicity-induced nonalcoholic steatohepatitis by regulating macrophage migration and M1/M2 status

Background and objectivesChemokine (C-X3-C motif) ligand 1 (CX3CL1) and its receptor CX3CR1 regulate the migration and activation of immune cells and are involved in the pathogenesis of nonalcoholic steatohepatitis (NASH), but the mechanism remains elusive. Here, the roles of CX3CL1/CX3CR1 in the macrophage migration and polarization in the livers of NASH mice were investigated. Methods and resultsThe expression of Cx3cl1 and Cx3cr1 was markedly upregulated in the livers of lipotoxicity-induced NASH mice. CX3CR1 was predominantly expressed by F4/80+ macrophages and to a lesser degree by hepatic stellate cells or endothelial cells in the livers of NASH mice. Flow cytometry analysis revealed that, compared with chow-fed mice, NASH mice exhibited a significant increase in CX3CR1+ expression by liver macrophages (LMs), particularly M1 LMs. CX3CR1 deficiency caused a significant increase in inflammatory monocyte/macrophage infiltration and a shift toward M1 dominant macrophages in the liver, thereby exacerbating the progression of NASH. Moreover, transplantation of Cx3cr1−/− bone marrow was sufficient to cause glucose intolerance, inflammation, and fibrosis in the liver. In addition, deletion of CCL2 in Cx3cr1−/− mice alleviated NASH progression by decreasing macrophage infiltration and inducing a shift toward M2 dominant LMs. Importantly, overexpression of CX3CL1 in vivo protected against hepatic fibrosis in NASH. ConclusionPharmacological therapy targeting liver CX3CL1/CX3CR1 signaling might be a candidate for the treatment of NASH.

CX3CR1 deficiency aggravates amyloid driven neuronal pathology and cognitive decline in Alzheimer’s disease

BackgroundDespite its identification as a key checkpoint regulator of microglial activation in Alzheimer’s disease, the overarching role of CX3CR1 signaling in modulating mechanisms of Aβ driven neurodegeneration, including accumulation of hyperphosphorylated tau is not well understood.MethodologyAccumulation of soluble and insoluble Aβ species, microglial activation, synaptic dysregulation, and neurodegeneration is investigated in 4- and 6-month old 5xFAD;Cx3cr1+/+ and 5xFAD;Cx3cr1−/− mice using immunohistochemistry, western blotting, transcriptomic and quantitative real time PCR analyses of purified microglia. Flow cytometry based, in-vivo Aβ uptake assays are used for characterization of the effects of CX3CR1-signaling on microglial phagocytosis and lysosomal acidification as indicators of clearance of methoxy-X-04+ fibrillar Aβ. Lastly, we use Y-maze testing to analyze the effects of Cx3cr1 deficiency on working memory.ResultsDisease progression in 5xFAD;Cx3cr1−/− mice is characterized by increased deposition of filamentous plaques that display defective microglial plaque engagement. Microglial Aβ phagocytosis and lysosomal acidification in 5xFAD;Cx3cr1−/− mice is impaired in-vivo. Interestingly, Cx3cr1 deficiency results in heighted accumulation of neurotoxic, oligomeric Aβ, along with severe neuritic dystrophy, preferential loss of post-synaptic densities, exacerbated tau pathology, neuronal loss and cognitive impairment. Transcriptomic analyses using cortical RNA, coupled with qRT-PCR using purified microglia from 6 month-old mice indicate dysregulated TGFβ-signaling and heightened ROS metabolism in 5xFAD;Cx3cr1−/− mice. Lastly, microglia in 6 month-old 5xFAD;Cx3cr1−/− mice express a ‘degenerative’ phenotype characterized by increased levels of Ccl2, Ccl5, Il-1β, Pten and Cybb along with reduced Tnf, Il-6 and Tgfβ1 mRNA.ConclusionsCx3cr1 deficiency impairs microglial uptake and degradation of fibrillar Aβ, thereby triggering increased accumulation of neurotoxic Aβ species. Furthermore, loss of Cx3cr1 results in microglial dysfunction typified by dampened TGFβ-signaling, increased oxidative stress responses and dysregulated pro-inflammatory activation. Our results indicate that Aβ-driven microglial dysfunction in Cx3cr1−/− mice aggravates tau hyperphosphorylation, neurodegeneration, synaptic dysregulation and impairs working memory.

CXCR4 blockade in macrophage promotes angiogenesis in ischemic hindlimb by modulating autophagy

Chemokine receptor CXCR4 plays a crucial role in leukocyte recruitment and inflammation regulation to influence tissue repair in ischemic diseases. Here we assessed the effect of CXCR4 expression in macrophages on angiogenesis in the ischemic hindlimb of a mouse. Inflammatory cells were increased in the ischemic muscles of hindlimb, and CXCR4 was highly expressed in the infiltrated macrophages but not in neutrophils. Myeloid-specific CXCR4 knockout attenuated macrophage infiltration and subsequent reduced inflammatory response in the ischemic hindlimb, accompanied with better blood reperfusion and higher capillary density as compared with that in LysM Cre+/− (Cre) mice. Similar outcomes were also observed in CRE mice whose bone marrow cells were replaced with those from CXCR4-deficient mice. Gene ontology cluster analysis reviewed that Decorin, a negative regulator of angiogenesis, was reduced in CXCR4-deficient macrophages. CXCR4-deficient macrophages were less inducible into M1 phase by lipopolysaccharide and more favorable for M2 polarization under oxygen/glucose deprivation condition. Enhanced autophagy was detected in CXCR4-deficient macrophages, which was associated with less expression of both Decorin and the inflammatory cytokines. In summary, myeloid-specific CXCR4 deficiency reduced monocyte infiltration and the secretion of inflammatory cytokines and Decorin from macrophages, thus blunting inflammation response and promoting angiogenesis in the ischemic hindlimb.

NLRP3 Inflammasome Is Involved in Cocaine-Mediated Potentiation on Behavioral Changes in CX3CR1-Deficient Mice.

Microglia, the primary immunocompetent cells of the brain, are suggested to play a role in the development of drug addiction. Previous studies have identified the microglia-derived pro-inflammatory factor IL1β can promote the progression of cocaine addiction. Additionally, the activation status of microglia and “two-hit hypothesis” have been proposed in the field of drug addiction to explain how early life stress (ELS) could significantly increase the incidence of drug addiction in later life. However, the mechanisms underlying microglia prime and full activation and their roles in drug addiction remain greatly unexplored. Here, we employed CX3CR1-GFP mice (CX3CR1 functional deficiency, CX3CR1−/−) to explore whether primed microglia could potentiate cocaine-mediated behavioral changes and the possible underlying mechanisms. CX3CR1−/− mice revealed higher hyperlocomotion activity and conditional place preference than wild-type (WT) mice did under cocaine administration. In parallel, CX3CR1−/− mice showed higher activity of NLR family pyrin domain-containing 3 (NLRP3) inflammasome than WT mice. Interestingly, CX3CR1 deficiency itself could prime NLRP3 signaling by increasing the expression of NLPR3 and affect lysosome biogenesis under basal conditions. Taken together, our findings demonstrated that the functional status of microglia could have an impact on cocaine-mediated reward effects, and NLRP3 inflammasome activity was associated with this phenomenon. This study was consistent with the two-hit hypothesis and provided solid evidence to support the involvement of microglia in drug addiction. Targeting the NLRP3 inflammasome may represent a novel therapeutic approach for ameliorating or blocking the development of drug addiction.

In vivo two-photon characterization of tumor-associated macrophages and microglia (TAM/M) and CX3CR1 during different steps of brain metastasis formation from lung cancer.

Brain metastases frequently occur in lung cancer and dramatically limit prognosis of affected patients. The influence of tumor-associated macrophages and microglia (TAM/M) and their receptor CX3CR1 on different steps of brain metastasis formation from lung cancer is poorly characterized. We established a syngeneic orthotopic cerebral metastasis model in mice by combining a chronic cranial window with repetitive intravital 2-photon laser scanning microscopy. This allowed in vivo tracking of fluorescence-expressing tumor cells and TAM/M on a single-cell level over weeks. Intracarotid injection of red tdTomato-fluorescent Lewis lung carcinoma cell was performed in transgenic mice either proficient or deficient for CX3CR1. After intracarotid cell injection, intravascular tumor cells extravasated into the brain parenchyma and formed micro- and mature macrometastases. We observed potential phagocytosis of extravasated tumor cells by TAM/M. However, during later steps of metastasis formation, these anti-tumor effects diminished and were paralleled by TAM/M accumulation and activation. Although CX3CR1 deficiency resulted in a lower number of extravasated tumor cells, progression of these extravasated cells into micro metastases was more efficient. Overall, this resulted in a comparable number of mature macrometastases in CX3CR1-deficient and -proficient mice. Our findings indicate that unspecific inhibition of CX3CR1 might not be a suitable therapeutic option to prevent dissemination of lung cancer cells to the brain. Given the close interaction between TAM/M and tumor cells during metastasis formation, other therapeutic approaches targeting TAM/M function may warrant further evaluation. The herein established orthotopic mouse model may be a useful tool to evaluate such concepts in vivo.

Roles of CX3CR1 and probiotic lactobacilli on the development of autoimmunity in MRL/lpr mice

Abstract Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease with no known cure. Gut microbiota and the immune system have a close relationship that controls inflammation. Alterations in the gut microbiota, known as dysbiosis, can trigger SLE in both human and mouse. We previously published differences between the gut microbiota in lupus-prone MRL/Mp-Faslpr (lpr) mice compared to healthy controls, with lower Lactobacillaceae abundance in lpr mice. Restoring the gut microbiota with a mix of 5 different strains of this family significantly attenuated lupus-liked disease in lpr mice. In this study, we sought to understand the potential benefit of this mix in lpr mice with Cx3cr1 deficiency (KO). KO mice on C57BL/6 background display increased translocation of commensal bacteria into the mesenteric lymph node (MLN) and increased susceptibility to intestinal inflammation. However, whether CX3CR1+ macrophages play a role in controlling systemic inflammation was unclear. After backcrossing the KO allele to lpr mice, we found that KO lpr mice exhibited exacerbated splenomegaly and lymphadenopathy, as well as increased proteinuria. Interestingly, treating the KO lpr mice with the 5 Lactobacillus strains attenuated the enlargement of MLN and abrogated the increase of proteinuria. Furthermore, the effects of the probiotic bacteria were accompanied by downregulation of serum endotoxin, suggesting the reversal of a leaky gut that may explain their benefits on KO lpr mice. Together, our results show an important role of CX3CR1+ macrophages in controlling systemic inflammation in lupus, and that probiotic Lactobacillus spp. may be an effective treatment against SLE through preventing bacterial translocation.

CX3CR1 deficiency aggravates brain white matter injury and affects expression of the CD36/15LO/NR4A1 signal

ObjectiveTo study the effects of CX3CR1 on white matter injury, neurofunction, recognition, and expression of the CD36/15LO/NR4A1 signal in mice with traumatic brain injury (TBI). MethodsCX3CR1GFP/GFP, CX3CR1GFP/+ and C57BL/6 male mice were randomly divided into 3 groups. We used a controlled cortical impact (CCI) to establish a TBI model and T2wt MRI to detect the TBI lesion. FA and DTI allowed for quantitative evaluation of the structural integrity of white matter tracts. Several behavior tests were used to investigate nerve function; a computer-based tracing system was used to trace and analyze dendrites and cell bodies of microglia and astrocytes in the peri-lesional brain areas. We also used RT-PCR and western blot to detect the effect of CX3CL1/CX3CR1 axis on CD36/15LO/NR4A1 signal. ResultsThe fractional anisotropy (FA) at the corpus callosum area of brain was decreased at 3 days post TBI, the average lesion volume CX3CR1GFP/GFP group was increased, and the neurologic deficit scores of mice of Cx3Cr1GFP/+ and wild-type groups were significantly increased compared to Cx3Cr1GFP/GFP group mice. In the Corner turn test, TBI induced impairments in forelimb function that were more severe than Cx3Cr11GFP/+ and wild-type TBI mice. We operated the Y-maze at 3 days post-TBI and the NOR test at 28 days after TBI. There was a significant TBI effect induced in decreased percentage entries into the novel arm in Cx3Cr1GFP/+ and wild-type TBI mice, compared with Cx3Cr1GFP/GFP; Cx3Cr1GFP/+. Wild-type mice showed decreased exploration time in new objects compared with Cx3Cr1GFP/GFP. Those two behavior tests demonstrated that Cx3Cr1 knock-out increased the damage caused by TBI to memory. In the tail suspension and force swimming tests, there was no significant difference between those three groups. CD36 increased in Cx3Cr1GFP/GFP compared with the other three groups at 3 days after TBI. TBI inhibited the expression of NR4A1 at 3 d after damage. Cx3Cr1 deficiency can induce high expression of 15LO, this was unaffected by TBI. ConclusionCX3CR1 deletion can enhance white matter injury. It increased the expression of CD36 and 15LO and increased expression of NR4A1. The lack of CX3CR1 can affect the recovery of nerve function.

CX3CR1 Depletion Promotes the Formation of Platelet-Neutrophil Complexes and Aggravates Acute Peritonitis.

Peritonitis is a life-threatening condition on intensive care units. Inflammatory cytokines and their receptors drive inflammation, cause the formation of platelet-neutrophil complexes (PNCs) and therefore the migration of polymorphonuclear neutrophils (PMNs) into the inflamed tissue. CX3CL1 and its receptor CX3CR1 are expressed in various cells, and promote inflammation. The shedding of CX3CL1 is mediated by a disintegrin and metalloprotease (ADAM) 17. The role of the CX3CL1-CX3CR1 axis in acute peritonitis remains elusive. In zymosan-induced peritonitis, we determined the formation of PNCs in the blood and the expression of PNC-related molecules on PNCs. PMN migration into the peritoneal lavage was evaluated in wild-type (WT) and CX3CR1-/- animals by flow cytometry. CX3CL1, ADAM17, and the expression of various inflammatory cytokines were detected. Further, we determined the inflammation-associated activation of the intracellular transcription factor extracellular signal-regulated kinase 1/2 (ERK1/2) by Western blot. The PMN accumulation in the peritoneal lavage and the PNC formation in the circulation were significantly raised in CX3CR1-/- compared with WT animals. The expression of PNC-related selectins on PNCs was significantly increased in the blood of CX3CR1-/- animals, as well as cytokine levels. Further, we observed an increased activation of ERK1/2 and elevated ADAM17 expression in CX3CR1-/- during acute inflammation. Selective ERK1/2 inhibition ameliorated inflammation-related increased ADAM17 expression. A CX3CR1 deficiency raised the release of inflammatory cytokines and increased the PNC formation respectively PMN migration via an elevated ERK1/2 activation during acute peritonitis. Further, we observed a link between the ERK1/2 activation and an elevated ADAM17 expression on PNC-related platelets and PMNs during inflammation. Our data thus illustrate a crucial role of CX3CR1 on the formation of PNCs and regulating inflammation in acute peritonitis.

Liver Immune Profiling Reveals Pathogenesis and Therapeutics for Biliary Atresia

Biliary atresia (BA) is a severe cholangiopathy that leads to liver failure in infants, but its pathogenesis remains to be fully characterized. By single-cell RNA profiling, we observed macrophage hypo-inflammation, Kupffer cell scavenger function defects, cytotoxic Tcell expansion, and deficiency of CX3CR1+effector T and natural killer (NK) cells in infants with BA. More importantly, we discovered that hepatic B cell lymphopoiesis did not cease after birth and that tolerance defects contributed to immunoglobulin G (IgG)-autoantibody accumulation in BA. In a rhesus-rotavirus induced BA model, depleting B cells or blocking antigen presentation ameliorated liver damage. In a pilot clinical study, we demonstrated that rituximab was effective in depleting hepatic B cells and restoring the functions of macrophages, Kupffer cells, and Tcells to levels comparable to those of control subjects. In summary, our comprehensive immune profiling in infants with BA had educed that B-cell-modifying therapies may alleviate liver pathology.

TAMI-02. ALTERATIONS IN C-X3-C MOTIF CHEMOKINE RECEPTOR 1 (CX3CR1) EXPRESSION INFLUENCE MICROGLIAL AND MACROPHAGE RESPONSE IN DIFFERENT STEPS OF CEREBRAL METASTASIS FORMATION

Abstract Metastasis to the brain is a frequent complication in lung cancer and is still associated with a dismal prognosis. Current treatment strategies not only target tumor cells but also focus on cells of the tumor microenvironment like tumor associated microglia/macrophages (TAMs). The interactions between tumor cells and TAMs during different steps of cerebral metastasis formation of lung cancer brain metastasis are poorly characterized. Moreover, the role of CX3CR1 in this process remains unclear. We established a syngeneic cerebral metastasis mouse model by combining a chronic cranial window and two-photon laser scanning microscopy (TPLSM), which allows the tracking of single fluorescent metastasizing tumor cells and the tumor microenvironment on a cellular resolution in vivoover time for a period of weeks. Transgenic CX3CR1 proficient and deficient mice (CX3CRGFP/wt and CX3CR1GFP/GFP) were injected with red fluorescent Lewis lung carcinoma cells. During different steps of metastasis formation (extravasation, formation of micro- and macrometastasis) the density and cell body volume of TAMs, their interaction with tumor cells and possible influence on the fate of single metastatic tumor cells were investigated using serial TPLSM. We found that during metastasis formation TAM density was significantly lower in CX3CR1 deficient mice. However, activation as assessed by TAM morphology did not differ in the absence of CX3CR1. Strikingly, CX3CR1 deficiency was associated with a significant increase of tumor cells successfully extravasating the cerebral vasculature. However, subsequent steps (mirco- and macrometastasis formation) were observed less frequent in CX3CR1 deficient mice. In summary, our results highlight a complex role of CX3CR1 for TAMs during cerebral metastasis formation, indicating anti-tumorous properties of CX3CR1 at early steps and possible pro-tumorous effects at later stages (micro- and macrometastasis formation).

Analysis of CX3CR1 haplodeficiency in male and female APPswe/PSEN1dE9 mice along Alzheimer disease progression

Microglia, the resident immune cells of the brain, have recently emerged as key players in Alzheimer Disease (AD) pathogenesis, but their roles in AD remain largely elusive and require further investigation. Microglia functions are readily altered when isolated from their brain environment, and microglia reporter mice thus represent valuable tools to study the contribution of these cells to neurodegenerative diseases such as AD. The CX3CR1+/eGFP mice is one of the most popular microglia reporter mice, and has been used in numerous studies to investigate in vivo microglial functions, including in the context of AD research. However, until now, the impact of CX3CR1 haplodeficiency on the typical features of Alzheimer Disease has not been studied in depth.To fill this gap, we generated APPswe/PSEN1dE9:CX3CR1+/eGFP mice and analyzed these mice for Alzheimer’s like pathology and neuroinflammation hallmarks. More specifically, using robust multifactorial statistical and multivariate analyses, we investigated the impact of CX3CR1 deficiency in both males and females, at three typical stages of the pathology progression: at early stage when Amyloid-β (Aβ) deposition just starts, at intermediate stage during Aβ accumulation phase and at more advanced stages when Aβ plaque number stabilizes. We found that CX3CR1 haplodeficiency had little impact on the progression of the pathology in the APPswe/PSEN1dE9 model and demonstrated that the APPswe/PSEN1dE9:CX3CR1+/eGFP line is a relevant and useful model to study the role of microglia in Alzheimer Disease. In addition, although Aβ plaques density is higher in females compared to age-matched males, we show that their glial reaction, inflammation status and memory deficits are not different.

CX3CR1 Deficiency Attenuates DNFB-Induced Contact Hypersensitivity Through Skewed Polarization Towards M2 Phenotype in Macrophages.

CX3CL1 can function as both an adhesion molecule and a chemokine for CX3CR1+ cells, such as T cells, monocytes, and NK cells. Recent studies have demonstrated that CX3CL1–CX3CR1 interaction is associated with the development of various inflammatory skin diseases. In this study, we examined CX3CR1 involvement in 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity using CX3CR1−/− mice. Ear swelling and dermal edema were attenuated after DNFB challenge in CX3CR1−/− mice. Expression of TNF-α, IL-6, and M1 macrophage markers was decreased in the ears of CX3CR1−/− mice, whereas expression of M2 macrophage markers including arginase-1 was increased. Decreased TNF-α and IL-6 expression and increased arginase-1 expression were found in peritoneal macrophages from CX3CR1−/− mice. Furthermore, ear swelling was attenuated by depleting dermal macrophages in wild-type mice to a similar level to CX3CR1−/− mice. These results suggest that CX3CR1 deficiency could induce skewed polarization towards M2 phenotype in macrophages, resulting in attenuation of contact hypersensitivity response.