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Background and Purpose: Because of the absence of effective therapies for metabolic dysfunction‐associated steatohepatitis (MASH), there is a rising interest in fibroblast growth factor 21 (FGF21) analogues due to their potential anti‐fibrotic activities in MASH treatment. PsTag‐FGF21, a long‐acting FGF21 analogue, has demonstrated promising therapeutic effects in several MASH mouse models. However, its efficacy and mechanism against MASH‐related fibrosis remain less well defined, compared with the specific mechanisms through which FGF21 improves glucose and lipid metabolism. Experimental Approach: The effectiveness of PsTag‐FGF21 was evaluated in two MASH‐fibrosis models. Co‐culture systems involving macrophages and hepatic stellate cells (HSCs) were employed for further assessment. Hepatic macrophages were selectively depleted by administering liposome‐encapsulated clodronate via tail vein injections. RNA sequencing and cytokine profiling were conducted to identify key factors involved in macrophage–HSC crosstalk. Key Results: We first demonstrated the significant attenuation of hepatic fibrosis by PsTag‐FGF21 in two MASH‐fibrosis models. Furthermore, we highlighted the crucial role of macrophage phenotypic switch in PsTag‐FGF21‐induced HSC deactivation. FGF21 was demonstrated to regulate macrophages in a PsTag‐FGF21‐like manner. NR4A1, a nuclear factor which is notably down‐regulated in human livers with MASH, was identified as a mediator responsible for PsTag‐FGF21‐induced phenotypic switch. Transcriptional control over insulin‐like growth factor 1, a crucial factor in macrophage–HSC crosstalk, was exerted by the intrinsically disordered region domain of NR4A1. Conclusion and Implications: Our results have elucidated the previously unclear mechanisms through which PsTag‐FGF21 treats MASH‐related fibrosis and identified NR4A1 as a potential therapeutic target for fibrosis. [ABSTRACT FROM AUTHOR]

Liver fibrosis is a disorder in which inflammatory reactions play an important role, and central to the progression and pathogenesis of this disease are the immune‐specific cells known as macrophages. Macrophage types are distinguished from each other by the expression of a series of surface markers. STAT6 and Arg1 play an important role in the polarization of macrophages, so these two factors are downstream of interleukin 4 (IL‐4) and IL‐13 cytokines and cause to differentiate M2. Therefore, this study aimed to compare the independent effects of imatinib and mesenchymal cell treatment on the polarization of macrophages in rat models of liver fibrosis. The liver fibrosis was induced by the injection of CCL4 for 6 weeks in Sprague–Dawley rats. Then, rats were divided into four different groups, and the effects of imatinib and mesenchymal cells on the expression of Arg1, Ly6c, and STAT6 were evaluated. Histopathology experiments considered the amelioration effect of treatments. Our results showed that Arg1 expression was significantly increased in the groups treated with mesenchymal cells and imatinib compared to the control group. On the other hand, expression of STAT6 was significantly increased in the imatinib‐treated mice compared to mesenchymal and control groups. Moreover, the expression of LY6C significantly decreased in imatinib and mesenchymal treated groups compared to the control group. Therefore, our data showed that mesenchymal stem cells and imatinib significantly modulate the fibrotic process in rat models of fibrosis, probably by polarizing macrophages towards an anti‐inflammatory profile and increasing the frequency of these cells in liver tissue. [ABSTRACT FROM AUTHOR]

A relatively high affinity/avidity of T cell receptor (TCR) recognition for self-peptide bound to major histocompatibility complex II (self-pMHC) ligands is a distinctive feature of CD4 T regulatory (Treg) cells, including their development in the thymus and maintenance of their suppressive functions in the periphery. Despite such high self-reactivity, however, all thymic-derived peripheral Treg populations are neither homogenous in their phenotype nor uniformly immune-suppressive in their function under steady state condition. We show here that based on the previously defined heterogeneity in the phenotype of peripheral Treg populations, Ly6C expression on Treg marks a lower degree of activation, proliferation, and differentiation status as well as functional incompetence. We also demonstrate that Ly6C expression on Treg in a steady state is either up- or downregulated depending on relative amounts of tonic TCR signals derived from its contacts with self-ligands. Interestingly, peripheral appearance and maintenance of these Ly6C-expressing Treg cells largely differed in an age-dependent manner, with their proportion being continuously increased from perinatal to young adult period but then being gradually declined with age. The reduction of Ly6C+ Treg in the aged mice was not due to their augmented cell death but rather resulted from downregulation of Ly6C expression. The Ly6C downregulation was accompanied by proliferation of Ly6C+ Treg cells and subsequent change into Ly6C- effector Treg with concomitant restoration of immune-suppressive activity. Importantly, we found that this phenotypic and functional change of Ly6C+ Treg is largely driven by conventional effector T cell population. Collectively, these findings suggest a potential cross-talk between peripheral Treg subsets and effector T cells and provides better understanding for Treg homeostasis and function on maintaining self-tolerance.

Background: The development of inflammatory bowel diseases is thought to be multifactorial, but the exact steps in pathogenesis are poorly understood. In this study, we investigated involvement of the activation of STAT1 signal pathway in the pathogenesis of an acute colitis model. Methods: A dextran sulfate sodium-induced acute colitis model was established by using wild-type C57BL/6 mice and STAT1-deficient mice. Disease indicators such as body weight loss and clinical score, induction of cytokines, chemokines, and inflammatory cells were evaluated in the acute colitis model. Results: Disease state was significantly improved in the acute colitis model using STAT1-deficient mice compared with wild-type mice. The induction of Ly6c-highly expressing cells in colorectal tissues was attenuated in STAT1-deficient mice. IL-6, CCL2, and CCR2 gene expressions in Ly6c-highly expressing cells accumulated in the inflamed colon tissues and were significantly higher than in Ly6c-intermediate-expressing cells, whereas TNF-α and IFN-α/β gene expression was higher in Ly6c-intermediate-expressing cells. Blockade of CCR2-mediated signaling significantly reduced the disease state in the acute colitis model. Conclusions: Two different types of Ly6c-expressing macrophages are induced in the inflamed tissues through the IFN-α/β-STAT1-mediated CCL2/CCR2 cascade and this is associated with the pathogenesis such as onset, exacerbation, and subsequent chronicity of acute colitis. [ABSTRACT FROM AUTHOR]

Peripheral circulating monocytes and resident macrophages are heterogeneous effector cells that play a critical role in the maintenance and restoration of pulmonary integrity. However, their detailed dynamic changes in lipopolysaccharide (LPS)-induced acute lung injury (ALI) remain unclear. Here, we investigated the impact of mononuclear phagocyte cells in the development of LPS-induced ALI/Acute respiratory distress syndrome (ARDS) and described the relations between the dynamic phenotypic changes and pulmonary pathological evolution. In this study, mice were divided into two groups and intraperitoneally injected with normal saline (NS) or LPS, respectively. A series of flow cytometry assay was performed for the quantification of peripheral circulating monocyte subpopulations, detection of the polarization state of bronchoalveolar lavage fluid (BALF)-isolated alveolar macrophages (AMϕ) and pulmonary interstitial macrophages (IMϕ) separated from lung tissues. Circulating Ly6Clo monocytes expanded rapidly after the LPS challenge on day 1 and then decreased to day 7, while Ly6Chi monocytes gradually increased and returned to normal level on the 7th day. Furthermore, the expansion of M2-like AMϕ (CD64+CD206+) was peaked on day 1 and remained high on the third day, while the polarization state of IMϕ (CD64+ CD11b+) was not influenced by the LPS challenge at all the time points. Taken together, our findings show that Ly6Clo monocytes and M2-like AMϕ form the major peripheral circulation and pulmonary immune cell populations, respectively. The dynamic changes of mononuclear phagocyte in three compartments after the LPS challenge may provide novel protective strategies for mononuclear phagocytes.

Osteoclasts (OCs) are bone‐resorbing cells formed by the serial fusion of monocytes. In mice and humans, three distinct subsets of monocytes exist; however, it is unclear if all of them exhibit osteoclastogenic potential. Here we show that in wild‐type (WT) mice, Ly6Chi and Ly6Cint monocytes are the primary source of OC formation when compared to Ly6C− monocytes. Their osteoclastogenic potential is dictated by increased expression of signaling receptors and activation of preestablished transcripts, as well as de novo gain in enhancer activity and promoter changes. In the absence of interferon regulatory factor 8 (IRF8), a transcription factor important for myelopoiesis and osteoclastogenesis, all three monocyte subsets are programmed to display higher osteoclastogenic potential. Enhanced NFATc1 nuclear translocation and amplified transcriptomic and epigenetic changes initiated at early developmental stages direct the increased osteoclastogenesis in Irf8‐deficient mice. Collectively, our study provides novel insights into the transcription factors and active cis‐regulatory elements that regulate OC differentiation. © 2020 American Society for Bone and Mineral Research (ASBMR). [ABSTRACT FROM AUTHOR]

Although CD4+ T cell memory is a critical component of adaptive immunity, antigen-specific CD4+ T cell recall responses to secondary infection have been inadequately studied. Here we examine the kinetics of the secondary response in an important immunological model, infection with attenuated Listeria monocytogenes (Lm). We identify CD4+ T cell subsets that preferentially expand during a secondary response and highlight the importance of prime-boost strategies in expanding and maintaining antigen-specific, tissue-resident memory CD4+ T cells. Following intravenous infection with an attenuated strain of Lm, we found that total antigen-specific CD4+ T cells responded more robustly in secondary compared with primary infection, reaching near-peak levels in secondary lymphoid organs (SLOs) and the liver by three days post-infection. During the secondary response, CD4+ T cells also contracted more quickly. Primary Lm infection generated two main classes of effector cells: Th1 cells that assist macrophages and T follicular helper (Tfh) cells that aid B cells in antibody production. We found that during the secondary response, a population of Ly6C+ Tfh cells emerged in SLOs and was the basis for the skewing of this response to a Tfh phenotype. Deletion of T-bet in T cells precluded development of Ly6C+ Tfh cells, but did not alter anti-Lm antibody responses. Moreover, during recall responses, CD49a+ Th1 cells preferentially expanded and accumulated in the liver, achieving a new set point. Parabiosis experiments indicated that, in contrast to Tfh cells and most splenic Th1 cells, the majority of CD49a+ Th1 cells in the liver were tissue resident. Overall, these data demonstrate a robust secondary CD4+ T cell response that differs in kinetics and composition from the primary response and provide insight into targets to enhance both peripheral and tissue-resident CD4+ T cell responses.

Although CD4+ T cell memory is a critical component of adaptive immunity, antigen-specific CD4+ T cell recall responses to secondary infection have been inadequately studied. Here we examine the kinetics of the secondary response in an important immunological model, infection with attenuated Listeria monocytogenes (Lm). We identify CD4+ T cell subsets that preferentially expand during a secondary response and highlight the importance of prime-boost strategies in expanding and maintaining antigen-specific, tissue-resident memory CD4+ T cells. Following intravenous infection with an attenuated strain of Lm, we found that total antigen-specific CD4+ T cells responded more robustly in secondary compared with primary infection, reaching near-peak levels in secondary lymphoid organs (SLOs) and the liver by three days post-infection. During the secondary response, CD4+ T cells also contracted more quickly. Primary Lm infection generated two main classes of effector cells: Th1 cells that assist macrophages and T follicular helper (Tfh) cells that aid B cells in antibody production. We found that during the secondary response, a population of Ly6C+ Tfh cells emerged in SLOs and was the basis for the skewing of this response to a Tfh phenotype. Deletion of T-bet in T cells precluded development of Ly6C+ Tfh cells, but did not alter anti-Lm antibody responses. Moreover, during recall responses, CD49a+ Th1 cells preferentially expanded and accumulated in the liver, achieving a new set point. Parabiosis experiments indicated that, in contrast to Tfh cells and most splenic Th1 cells, the majority of CD49a+ Th1 cells in the liver were tissue resident. Overall, these data demonstrate a robust secondary CD4+ T cell response that differs in kinetics and composition from the primary response and provide insight into targets to enhance both peripheral and tissue-resident CD4+ T cell responses. [ABSTRACT FROM AUTHOR]

• Circulating monocytes present a pro-inflammatory profile and phenotype in obesity. • In obesity, β2 adrenergic stimulation exerts anti-inflammatory effects on monocytes. • β2 adrenergic activation exerts pro-inflammatory effects on monocytes in lean mice. • β2 adrenergic regulation of inflammation depends on inflammatory basal set-point. Obesity is a chronic condition associated with low-grade inflammation, and it also involves alterations of the function of the hypothalamic-pituitaryadrenal axis and the sympathetic nervous system. Adrenergic agonists such as catecholamines are important immunoregulatory molecules that are involved in modulating both metabolism and most of the mechanisms of the immune response. The first objective of this study was to determine whether the systemic inflammatory state associated with obesity is also manifested in the inflammatory profile and phenotype of circulating monocytes; and the second objective was to evaluate the effects of β2 adrenergic stimulation on the inflammatory profile and phenotype of monocytes in obesity, and whether this response could be different from that in lean individuals. C57BL/6J mice were randomly allocated to one of two diets for 18 weeks: high-fat diet in order to obtain an experimental model of obesity, and standard diet in the control lean group. Circulating monocyte expression of inflammatory cytokines (MCP-1, TNF-α, IL-8, IL-6, IL-10, and TGF-β), surface membrane marker Ly6C, inducible nitric oxide synthase and arginase-1, and Toll-like receptor 4 were evaluated through flow cytometry in the presence or absence of selective β2 adrenergic receptor agonist terbutaline. Monocytes from high-fat diet-induced obese animals presented higher expression levels of all pro-inflammatory cytokines and a higher percentage of monocytes with a pro-inflammatory phenotype than those from lean animals. β2 adrenergic stimulation induced a shift towards an anti-inflammatory activity profile and phenotype in obese mice, whereas it induced a shift towards a pro-inflammatory activity profile and phenotype in lean mice. In conclusion, β2 adrenergic stimulation in monocytes was anti-inflammatory only in obese animals, which presented a pro-inflammatory state at baseline. [ABSTRACT FROM AUTHOR]

Background/Aims: Inflammatory processes are controlled by the fine-tuned balance of monocyte subsets. In mice, different subsets of monocytes can be distinguished by the expression of Ly6C that is highly expressed on inflammatory monocytes (Ly6Chigh) and to a lesser extent on patrolling monocytes (Ly6Clow). Our previous study revealed an accumulation of Ly6Chigh monocytes in atherosclerotic-prone mice bearing a deficiency in suppressor of cytokine signaling (SOCS)-1 leading to an increased atherosclerotic burden. To decipher the underlying mechanisms, we performed a genome-wide analysis of SOCS-1-dependent gene regulation in Ly6Chigh and Ly6Clow monocytes. Methods: In monocyte subsets from SOCS-1- competent and -deficient mice differentially regulated genes were identified using an Illumina mRNA microarray (45,200 transcripts), which were randomly validated by qPCR. Principal component analysis was performed to further characterize mRNA profiles in monocyte subsets. To unravel potential regulatory mechanisms behind the differential mRNA expression, in silico analysis of a transcription factor (TF) network correlating with SOCS-1-dependent mRNA expression was carried out and combined with a weighted correlation network analysis (WGCNA). Results: mRNA analysis in monocyte subsets revealed 46 differentially regulated genes by 2-fold or more. Principal component analysis illustrated a distinct separation of mRNA profiles in monocyte subsets from SOCS-1-deficient mice. Notably, two cell surface receptors crucially involved in the determination of monocyte differentiation and survival, C-X3-C chemokine receptor 1 (CX3CR1) and colony stimulating factor 1 receptor (CSF1R), were identified to be regulated by SOCS-1. Moreover, in silico analysis of a TF network in combination with the WGCNA revealed genes coding for PPAR-γ, NUR77 and several ETSdomain proteins that act as pivotal inflammatory regulators. Conclusion: Our study reveals that SOCS-1 is implicated in a TF network regulating the expression of central transcription factors like PPAR-γ and NUR77 thereby influencing the expression of CX3CR1 and CSF1R that are known to be pivotal for the survival of Ly6Clow monocytes. [ABSTRACT FROM AUTHOR]

Abstract Monocytes are important mediators of the innate immunity by recognizing and attacking especially bacterial pathogens but also play crucial roles in various inflammatory diseases, including vascular inflammation and atherosclerosis. Maturation, differentiation and function of monocytes have been intensively explored for a long time in innumerable experimental and clinical studies. Monocytes do not represent a uniform cell type but could be further subdivided into subpopulations with distinct features and functions. Those subpopulations have been identified in experimental mouse models as well as in humans, albeit distinguished by different cell surface markers. While Ly6C is used for subpopulation differentiation in mice, corresponding human subsets are differentiated by CD14 and CD16. In this review, we specifically focused on new experimental insights from recent years mainly in regard to murine monocyte subpopulations and their roles in vascular inflammation und atherogenesis. Graphical abstract Unlabelled Image [ABSTRACT FROM AUTHOR]

A relatively high affinity/avidity of T cell receptor (TCR) recognition for self-peptide bound to major histocompatibility complex II (self-pMHC) ligands is a distinctive feature of CD4 T regulatory (Treg) cells, including their development in the thymus and maintenance of their suppressive functions in the periphery. Despite such high self-reactivity, however, all thymic-derived peripheral Treg populations are neither homogenous in their phenotype nor uniformly immune-suppressive in their function under steady state condition. We show here that based on the previously defined heterogeneity in the phenotype of peripheral Treg populations, Ly6C expression on Treg marks a lower degree of activation, proliferation, and differentiation status as well as functional incompetence. We also demonstrate that Ly6C expression on Treg in a steady state is either up- or downregulated depending on relative amounts of tonic TCR signals derived from its contacts with self-ligands. Interestingly, peripheral appearance and maintenance of these Ly6C-expressing Treg cells largely differed in an age-dependent manner, with their proportion being continuously increased from perinatal to young adult period but then being gradually declined with age. The reduction of Ly6C+ Treg in the aged mice was not due to their augmented cell death but rather resulted from downregulation of Ly6C expression. The Ly6C downregulation was accompanied by proliferation of Ly6C+ Treg cells and subsequent change into Ly6C− effector Treg with concomitant restoration of immune-suppressive activity. Importantly, we found that this phenotypic and functional change of Ly6C+ Treg is largely driven by conventional effector T cell population. Collectively, these findings suggest a potential cross-talk between peripheral Treg subsets and effector T cells and provides better understanding for Treg homeostasis and function on maintaining self-tolerance.

Monocytosis is considered a poor prognostic factor for many cancers, including B cell lymphomas. The mechanisms by which different monocyte subsets support the growth of lymphoma is poorly understood. Using a pre-clinical mouse model of B cell non-Hodgkin's lymphoma (B-NHL), we investigated the impact of tumor progression on circulating monocyte levels, subset distribution and their activity, with a focus on immune suppression. B-NHL development corresponded with significant expansion initially of classical (Ly6Chi) and non-classical (Ly6Clo) monocytes, with accumulation and eventual predominance of Ly6Clo cells. The lymphoma environment promoted the conversion, preferential survival and immune suppressive activity of Ly6Clo monocytes. Ly6Clo monocytes expressed higher levels of immunosuppressive genes including PD-L1/2, Arg1, IDO1 and CD163, compared to Ly6Chi monocytes. Both monocyte subsets suppressed CD8 T cell proliferation and IFN-γ production in vitro, but via different mechanisms. Ly6Chi monocyte suppression was contact dependent, while Ly6Clo monocytes suppressed via soluble mediators, including IDO and arginase. Ly6Clo monocytes could be selectively depleted in tumor-bearing hosts by liposomal doxorubicin treatment, further enhanced by co-administration of anti-4-1BB monoclonal antibody. This treatment led to a reduction in tumor growth, but failed to improve overall survival. Analogous immunosuppressive monocytes were observed in peripheral blood of diffuse large B cell lymphoma patients and actively suppressed human CD8 T cell proliferation. This study highlights a potential immune evasion strategy deployed by B cell lymphoma involving accumulation of circulating non-classical monocytes with immunosuppressive activity.

A key event in liver fibrosis is the activation of the hepatic stellate cell (HSC). Schisandrin B (Sch B), a major component extracted from Schisandra chinensis , has been shown to inhibit HSC activation. Recently, ferroptosis (FPT) has been reported to be involved in HSC activation. However, whether Sch B has an effect on the HSC FPT remains unclear. Herein, we explored the effects of Sch B on liver fibrosis in vivo and in vitro and the roles of Wnt agonist 1 and ferrostatin-1 in the antifibrotic effects of Sch B. Sch B effectively alleviated CCl 4 -induced liver fibrosis, with decreased collagen deposition and α-SMA level. Additionally, Sch B resulted in an increase in lymphocyte antigen 6 complex locus C low (Ly6C lo ) macrophages, contributing to a reduced level of TIMP1 and increased MMP2. Notably, the Wnt pathway was involved in Sch B-mediated Ly6C macrophage phenotypic transformation. Further studies demonstrated that Sch B-treated macrophages had an inhibitory effect on HSC activation, which was associated with HSC FPT. GPX4, a negative regulator of FPT, was induced by Sch B and found to be involved in the crosstalk between macrophage and HSC FPT. Furthermore, HSC inactivation as well as FPT induced by Sch B-treated macrophages was blocked down by Wnt pathway agonist 1. Collectively, we demonstrate that Sch B inhibits liver fibrosis, at least partially, through mediating Ly6C lo macrophages and HSC FPT. Sch B enhances Wnt pathway inactivation, leading to the increase in Ly6C lo macrophages, which contributes to HSC FPT. Sch B may be a promising drug for liver fibrosis treatment.

Abstract: Myeloid‐derived suppressor cells (MDSCs) promote tumor growth through, in part, inhibiting T‐cell immunity. However, mechanisms underlying MDSC expansion and guidance of MDSCs toward the tumor microenvironment remain unclear. Employing Percoll density gradients, we separate bone marrow (BM) leukocytes from tumor‐bearing mice into four density‐increasing bands with myeloid leukocytes enriched in bands III and IV. Band III comprises monocytes and low‐density granulocytes, both confirmed to be M‐MDSCs and G‐MDSCs, respectively, by displaying potent inhibition of T‐cell proliferation. However, monocytes act as M‐MDSCs not only under tumor conditions but also the healthy condition. In contrast, band IV contains non‐inhibitory, mature granulocytes. Only band III G‐MDSCs display significant expansion in mice bearing B16 melanoma, Lewis lung carcinoma, or MC38 colon carcinoma. The expanded G‐MDSCs also show increased CXCR2 expression, which guides egress out of BM, and produce arginase‐1 and ROS upon encountering antigen‐activated T cells. Adoptive transfer assays demonstrate that both G‐MDSCs and mature granulocytes infiltrate tumors, but only the former displays sustention and accumulation. Intratumoral administrations of granulocytes further demonstrate that G‐MDSCs promote tumor growth, whereas mature granulocytes exert minimal effects, or execute powerful anti‐tumor effects providing the presence of PMN activation mechanisms in the tumor microenvironment. [ABSTRACT FROM AUTHOR]

Pyk2 expression is upregulated upon monocyte differentiation, and promotes apoptosis in mouse monocyte populations. Monocytes are short‐lived myeloid cells that perform functions essential for tissue homeostasis and disease resolution. However, the cellular mechanisms controlling the maintenance and turnover of monocyte populations are largely undefined. Proline‐rich tyrosine kinase 2 (Pyk2) is a nonreceptor tyrosine kinase that regulates numerous immune cell functions, but its role in monocytes is currently unknown. In this study, we sought to characterize the expression and function of Pyk2 in lineage‐committed monocyte populations. Here, we report that Pyk2 protein expression is increased in the Ly6C− monocyte population. Using a Pyk2 knockout mouse model (Pyk2−/−), we show that Pyk2 regulates the relative proportion of monocyte subsets normally represented in the bone marrow (BM) at steady state. In support of this conclusion, a similar phenotype was observed in the peripheral blood and spleen. Data from reciprocal BM chimera experiments indicate that the alterations in monocyte populations exhibited by Pyk2−/− mice are due to factors intrinsic to the monocytes. Lineage‐tracing of monocyte populations suggests that Pyk2 promotes apoptosis in BM monocytes, thereby acting as an important homeostatic regulator of turnover in these short‐lived, innate immune cells. [ABSTRACT FROM AUTHOR]

Background: Controversy exists regarding which cell types are responsible for autoantigen presentation in the retina during experimental autoimmune uveitis (EAU) development. In this study, we aimed to identify and characterize the retinal resident and infiltrating cells susceptible to express major histocompatibility complex (MHC) class II during EAU.Methods: EAU was induced in C57BL/6 mice by adoptive transfer of autoreactive lymphocytes from IRBP1-20-immunized animals. MHC class II expression was studied by immunostainings on eye cryosections. For flow cytometry (FC) analysis, retinas were dissected and enzymatically digested into single-cell suspensions. Three MHC class II+ retinal cell populations were sorted by FC, and their RNA processed for RNA-Seq.Results: Immunostainings demonstrate strong induction of MHC class II expression in EAU, especially in the inner retina at the level of inflamed vessels, extending to the outer retinal layers and the subretinal space in severely inflamed eyes. Most MHC class II+ cells express the hematopoietic marker IBA1. FC quantitative analyses demonstrate that MHC class II induction significantly correlates with disease severity and is associated with upregulation of co-stimulatory molecule expression. In particular, most MHC class IIhi cells express co-stimulatory molecules during EAU. Further phenotyping identified three MHC class II+ retinal cell populations: CD45-CD11b- non-hematopoietic cells with low MHC class II expression and CD45+CD11b+ hematopoietic cells with higher MHC class II expression, which can be further separated into Ly6C+ and Ly6C- cells, possibly corresponding to infiltrating macrophages and resident microglia. Transcriptome analysis of the three sorted populations leads to a clear sample clustering with some enrichment in macrophage markers and microglial cell markers in Ly6C+ and Ly6C- cells, respectively. Functional annotation analysis reveals that both hematopoietic cell populations are more competent in MHC class II-associated antigen presentation and in T cell activation than non-hematopoietic cells.Conclusion: Our results highlight the potential of cells of hematopoietic origin in local antigen presentation, whatever their Ly6C expression. Our work further provides a first transcriptomic study of MHC class II-expressing retinal cells during EAU and delivers a series of new candidate genes possibly implicated in the pathogenesis of retinal autoimmunity. [ABSTRACT FROM AUTHOR]

A series of antibodies against vascular endothelial growth factor (VEGF) have been developed for the treatment of various types of cancer, including non‑small cell lung cancer (NSCLC) in recent years. However, tumors frequently demonstrate resistance to these strategies of VEGF inhibition. Efforts to better understand the mechanism underlying the acquired resistance to anti‑VEGF antibodies are warranted. In the present study, in order to develop a xenograft model of acquired resistance to anti‑VEGF antibody, xenografts of human adenocarcinoma A549 cells were generated through the successive inoculation of tumor tissue explants into first (F1), second (F2) and third (F3) generations of mice treated with the anti‑VEGF antibody B20. Tumor growth rate and vessel‑forming ability, assessed via cluster of differentiation (CD) 31 staining, were significantly lower in the F1, F2 and F3 groups compared with in the F0 control group (P<0.01), suggesting that drug resistance was not successfully acquired. The percentages of CD11b+ myeloid‑derived suppressor cells and lymphocyte antigen 6C (Ly6C)+ subsets were significantly smaller in F1, F2 and F3 groups compared with in F0 (P<0.01). However, the ratio of Ly6C+ to CD11b+ cells was significantly higher in the F3 group compared with in F0 and F1 groups (P<0.01), indicating increasing recruitment of the Ly6C+ subset with successive challenges with the anti‑VEGF antibody. In conclusion, the recruitment of CD11b+Ly6C+ monocytes increased with successive generations of NSCLC‑xenografted mice challenged by B20, an anti‑VEGF agent. [ABSTRACT FROM AUTHOR]

Monocytes are central to the physiopathology of arthritis, but their roles in progression and resolution of the disease remain to be clarified. Using NR4A1−/− mice, which lack patrolling lymphocyte antigen 6C (Ly6Clow) monocytes, we found that inflammatory Ly6Chigh monocytes contribute to rapid development of arthritis in a serum transfer-induced arthritis (STIA) model. Our experiments suggest that patrolling monocytes do not promote the initiation and progression of arthritis in mice, as severity of symptoms was amplified in NR4A1−/− mice. Moreover, we show that treatment of arthritic wild type (WT) mice with cytosporone B (Csn-B), a NR4A1-specific agonist, significantly reduces severity of disease. Effects of Csn-B were absent in monocyte-depleted mice treated with clodronate until Ly6Clow monocytes were restored. Adoptive transfer of Ly6Clow monocytes in arthritic NR4A1−/− mice treated with Csn-B reduces joint inflammation, supporting the regulatory role of Ly6Clow subset on disease development. Our results also reveal that administration of Csn-B to arthritic mice enhances levels of circulating CD4+CD25+FoxP3+ Treg cells, a process requiring the presence of Ly6Clow monocytes. Together, these data indicate that Ly6Chigh monocytes are involved in the initiation and progression of arthritis and Ly6Clow monocytes contribute to reduce joint inflammation through the mobilization of Treg cells. [ABSTRACT FROM AUTHOR]

The formation of the atherosclerotic lesion is a complex process influenced by an array of inflammatory and lipid metabolism pathways. We previously demonstrated that NR4A nuclear receptors are highly induced in macrophages in response to inflammatory stimuli and modulate the expression of genes linked to inflammation in vitro. Here we used mouse genetic models to assess the impact of NR4A expression on atherosclerosis development and macrophage polarization. Transplantation of wild-type, Nur77−/−, or Nor1−/− null hematopoetic precursors into LDL receptor (LDLR)−/− recipient mice led to comparable development of atherosclerotic lesions after high-cholesterol diet. We also observed comparable induction of genes linked to M1 and M2 responses in wild-type and Nur77-null macrophages in response to lipopolysaccharides and interleukin (IL)-4, respectively. In contrast, activation of the nuclear receptor liver X receptor (LXR) strongly suppressed M1 responses, and ablation of signal transductor and activator of transcription 6 (STAT6) strongly suppressed M2 responses. Recent studies have suggested that alterations in levels of Ly6Clo monocytes may be a contributor to inflammation and atherosclerosis. In our study, loss of Nur77, but not Nor1, was associated with decreased abundance of Ly6Clo monocytes, but this change was not correlated with atherosclerotic lesion development. Collectively, our results suggest that alterations in the Ly6Clo monocyte population and bone marrow NR4A expression do not play dominant roles in macrophage polarization or the development of atherosclerosis in mice.

AbstractMonocytes are primitive hematopoietic cells that primarily arise from the bone marrow, circulate in the peripheral blood and give rise to differentiated macrophages. Over the past two decades, considerable attention to monocyte diversity and macrophage polarization has provided contextual clues into the role of myelomonocytic derivatives in human disease. Until recently, human monocytes were subdivided based on expression of the surface marker CD16. “Classical” monocytes express surface markers denoted as CD14++CD16− and account for greater than 70% of total monocyte count, while “non‐classical” monocytes express the CD16 antigen with low CD14 expression (CD14+CD16++). However, recognition of an intermediate population identified as CD14++CD16+ supports the new paradigm that monocytes are a true heterogeneous population and careful identification of specific subpopulations is necessary for understanding monocyte function in human disease. Comparative studies of monocytes in mice have yielded more dichotomous results based on expression of the Ly6C antigen. In this review, we will discuss the use of monocyte subpopulations as biomarkers of human disease and summarize correlative studies in mice that may yield significant insight into the contribution of each subset to disease pathogenesis.

The field of neuroimmunology is rapidly advancing. There is a growing appreciation for heterogeneity, both in inflammatory composition and region-specific inflammatory responses. This understanding underscores the importance of developing targeted immunomodulatory therapies for treating neurological disorders. Concerning neurotrauma, there is a dearth of publications directly comparing inflammatory responses in the brain and spinal cord after injury. The question therefore remains as to whether inflammatory cells responding to spinal cord vs. brain injury adopt similar functions and are therefore amenable to common therapies. In this review, we address this question while revisiting and modernizing the conclusions from publications that have directly compared inflammation across brain and spinal cord injuries. By examining molecular differences, anatomical variations, and inflammatory cell phenotypes between the injured brain and spinal cord, we provide insight into how neuroinflammation relates to neurotrauma and into fundamental differences between the brain and spinal cord. [ABSTRACT FROM AUTHOR]

Abstract: Pancreatic cancer is a highly fatal disease characterized by a dominant stroma formation. Exploring new biological targets, specifically those overexpressed in stroma cells, holds significant potential for the design of specific nanocarriers to attain homing of therapeutic and imaging agents to the tumor. In clinical specimens of pancreatic cancer, we found increased expression of CD59 in tumor associated endothelial cells as well as infiltrating cells in the stroma as compared to uninvolved pancreas. We explored this dual targeting effect using orthotopic human pancreatic cancer in nude mice. By immunofluorescence analysis, we confirmed the increased expression of Ly6C, mouse homolog of CD59, in tumor associated endothelial cells as well as in macrophages within the stroma. We decorated the surface of porous silicon nanocarriers with Ly6C antibody. Targeted nanocarriers injected intravenously accumulated to tumor associated endothelial cells within 15min. At 4h after administration, 9.8±2.3% of injected dose/g tumor of the Ly6C targeting nanocarriers accumulated in the pancreatic tumors as opposed to 0.5±1.8% with non-targeted nanocarriers. These results suggest that Ly6C (or CD59) can serve as a novel dual target to deliver therapeutic agents to the stroma of pancreatic tumors. [Copyright &y& Elsevier]

Peripheral circulating monocytes and resident macrophages are heterogeneous effector cells that play a critical role in the maintenance and restoration of pulmonary integrity. However, their detailed dynamic changes in lipopolysaccharide (LPS)-induced acute lung injury (ALI) remain unclear. Here, we investigated the impact of mononuclear phagocyte cells in the development of LPS-induced ALI/Acute respiratory distress syndrome (ARDS) and described the relations between the dynamic phenotypic changes and pulmonary pathological evolution. In this study, mice were divided into two groups and intraperitoneally injected with normal saline (NS) or LPS, respectively. A series of flow cytometry assay was performed for the quantification of peripheral circulating monocyte subpopulations, detection of the polarization state of bronchoalveolar lavage fluid (BALF)-isolated alveolar macrophages (AMϕ) and pulmonary interstitial macrophages (IMϕ) separated from lung tissues. Circulating Ly6C lo monocytes expanded rapidly after the LPS challenge on day 1 and then decreased to day 7, while Ly6C hi monocytes gradually increased and returned to normal level on the 7th day. Furthermore, the expansion of M2-like AMϕ (CD64 + CD206 + ) was peaked on day 1 and remained high on the third day, while the polarization state of IMϕ (CD64 + CD11b + ) was not influenced by the LPS challenge at all the time points. Taken together, our findings show that Ly6C lo monocytes and M2-like AMϕ form the major peripheral circulation and pulmonary immune cell populations, respectively. The dynamic changes of mononuclear phagocyte in three compartments after the LPS challenge may provide novel protective strategies for mononuclear phagocytes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Xiang, Peng and Ji.)

Abstract Background Neuropathology caused by acute viral infection of the brain is associated with the development of persistent neurological deficits. Identification of the immune effectors responsible for injuring the brain during acute infection is necessary for the development of therapeutic strategies that reduce neuropathology but maintain immune control of the virus. Methods The identity of brain-infiltrating leukocytes was determined using microscopy and flow cytometry at several acute time points following intracranial infection of mice with the Theiler's murine encephalomyelitis virus. Behavioral consequences of immune cell depletion were assessed by Morris water maze. Results Inflammatory monocytes, defined as CD45hiCD11b++F4/80+Gr1+1A8-, and neutrophils, defined as CD45hiCD11b+++F4/80-Gr1+1A8+, were found in the brain at 12 h after infection. Flow cytometry of brain-infiltrating leukocytes collected from LysM: GFP reporter mice confirmed the identification of neutrophils and inflammatory monocytes in the brain. Microscopy of sections from infected LysM:GFP mice showed that infiltrating cells were concentrated in the hippocampal formation. Immunostaining confirmed that neutrophils and inflammatory monocytes were localized to the hippocampal formation at 12 h after infection. Immunodepletion of inflammatory monocytes and neutrophils but not of neutrophils only resulted in preservation of hippocampal neurons. Immunodepletion of inflammatory monocytes also preserved cognitive function as assessed by the Morris water maze. Conclusions Neutrophils and inflammatory monocytes rapidly and robustly responded to Theiler's virus infection by infiltrating the brain. Inflammatory monocytes preceded neutrophils, but both cell types were present in the hippocampal formation at a timepoint that is consistent with a role in triggering hippocampal pathology. Depletion of inflammatory monocytes and neutrophils with the Gr1 antibody resulted in hippocampal neuroprotection and preservation of cognitive function. Specific depletion of neutrophils with the 1A8 antibody failed to preserve neurons, suggesting that inflammatory monocytes are the key effectors of brain injury during acute picornavirus infection of the brain. These effector cells may be important therapeutic targets for immunomodulatory or immunosuppressive therapies aimed at reducing or preventing central nervous system pathology associated with acute viral infection.

Although CD4 + T cell memory is a critical component of adaptive immunity, antigen-specific CD4 + T cell recall responses to secondary infection have been inadequately studied. Here we examine the kinetics of the secondary response in an important immunological model, infection with attenuated Listeria monocytogenes (Lm). We identify CD4 + T cell subsets that preferentially expand during a secondary response and highlight the importance of prime-boost strategies in expanding and maintaining antigen-specific, tissue-resident memory CD4 + T cells. Following intravenous infection with an attenuated strain of Lm, we found that total antigen-specific CD4 + T cells responded more robustly in secondary compared with primary infection, reaching near-peak levels in secondary lymphoid organs (SLOs) and the liver by three days post-infection. During the secondary response, CD4 + T cells also contracted more quickly. Primary Lm infection generated two main classes of effector cells: Th1 cells that assist macrophages and T follicular helper (Tfh) cells that aid B cells in antibody production. We found that during the secondary response, a population of Ly6C + Tfh cells emerged in SLOs and was the basis for the skewing of this response to a Tfh phenotype. Deletion of T-bet in T cells precluded development of Ly6C + Tfh cells, but did not alter anti-Lm antibody responses. Moreover, during recall responses, CD49a + Th1 cells preferentially expanded and accumulated in the liver, achieving a new set point. Parabiosis experiments indicated that, in contrast to Tfh cells and most splenic Th1 cells, the majority of CD49a + Th1 cells in the liver were tissue resident. Overall, these data demonstrate a robust secondary CD4 + T cell response that differs in kinetics and composition from the primary response and provide insight into targets to enhance both peripheral and tissue-resident CD4 + T cell responses., (Copyright © 2020 Malhotra, Burrack, Jenkins and Frosch.)

Candida auris has become a global public health threat due to its multidrug resistance and persistence. Currently, there are limited murine models to study C. auris infection. Those models use a combination of cyclophosphamide and cortisone acetate, suppressing both innate and adaptive immunity. Here, we compare C. auris infection in two neutrophil-depleted murine models in which innate immunity is targeted using the monoclonal antibodies 1A8 and RB6-8C5., (Copyright © 2020 American Society for Microbiology.)

Anomalous immune/inflammatory responses in obesity take place along with alterations in the neuroendocrine responses and dysregulation in the immune/stress feedback mechanisms. Exercise is a potential anti-inflammatory strategy in this context, but the influence of exercise on the β2 adrenergic regulation of the monocyte-mediated inflammatory response in obesity remains completely unknown. The first objective of this study was to analyze the effect of exercise on the inflammatory profile and phenotype of monocytes from obese and lean animals, and the second aim was to determine whether obesity could affect monocytes' inflammatory response to β2 adrenergic activation in exercised animals. C57BL/6J mice were allocated to different lean or obese groups: sedentary, with acute exercise, or with regular exercise. The inflammatory profile and phenotype of their circulating monocytes were evaluated by flow cytometry in the presence or absence of the selective β2 adrenergic receptor agonist terbutaline. Exercise caused an anti-inflammatory effect in obese individuals and a pro-inflammatory effect in lean individuals. β2 adrenergic receptor stimulation exerted a global pro-inflammatory effect in monocytes from exercised obese animals and an anti-inflammatory effect in monocytes from exercised lean animals. Thus, β2 adrenergic regulation of inflammation in monocytes from exercised animals seems to depend on the inflammatory basal set-point. [ABSTRACT FROM AUTHOR]

Two types of monocytes, Ly6Chi and Ly6Clo, infiltrate the heart in murine experimental autoimmune myocarditis (EAM). We discovered a role for cardiac fibroblasts in facilitating monocyte-to-macrophage differentiation of both Ly6Chi and Ly6Clo cells, allowing these macrophages to perform divergent functions in myocarditis progression. During the acute phase of EAM, IL-17A is highly abundant. It signals through cardiac fibroblasts to attenuate efferocytosis of Ly6Chi monocyte-derived macrophages (MDMs) and simultaneously prevents Ly6Clo monocyte-to-macrophage differentiation. We demonstrated an inverse clinical correlation between heart IL-17A levels and efferocytic receptor expressions in humans with heart failure (HF). In the absence of IL-17A signaling, Ly6Chi MDMs act as robust phagocytes and are less pro-inflammatory, whereas Ly6Clo monocytes resume their differentiation into MHCII+ macrophages. We propose that MHCII+Ly6Clo MDMs are associated with the reduction of cardiac fibrosis and prevention of the myocarditis sequalae. • Cardiac fibroblasts facilitate differentiation of Ly6Chi and Ly6Clo monocytes • IL-17A trans-signaling inhibits Ly6Clo monocyte-to-macrophage differentiation • IL-17A trans-signaling increases MerTK shedding on monocyte-derived macrophages • IL-17A trans-signaling promotes monocyte-derived macrophages to be proinflammatory Hou et al. show that cardiac fibroblasts facilitate infiltrating Ly6Chi and Ly6Clo monocytes to become macrophages. IL-17A trans-signaling through cardiac fibroblasts increases MerTK shedding and promotes a pro-inflammatory and pro-tissue remodeling gene expression profile in Ly6Chi monocyte-derived macrophages. Paradoxically, IL-17A signaling through cardiac fibroblasts can substantially inhibit Ly6Clo monocyte-to-macrophage differentiation. [ABSTRACT FROM AUTHOR]

Background/aims: Inflammatory processes are controlled by the fine-tuned balance of monocyte subsets. In mice, different subsets of monocytes can be distinguished by the expression of Ly6C that is highly expressed on inflammatory monocytes (Ly6C high ) and to a lesser extent on patrolling monocytes (Ly6C low ). Our previous study revealed an accumulation of Ly6C high monocytes in atherosclerotic-prone mice bearing a deficiency in suppressor of cytokine signaling (SOCS)-1 leading to an increased atherosclerotic burden. To decipher the underlying mechanisms, we performed a genome-wide analysis of SOCS-1-dependent gene regulation in Ly6C high and Ly6C low monocytes., Methods: In monocyte subsets from SOCS-1competent and -deficient mice differentially regulated genes were identified using an Illumina mRNA microarray (45,200 transcripts), which were randomly validated by qPCR. Principal component analysis was performed to further characterize mRNA profiles in monocyte subsets. To unravel potential regulatory mechanisms behind the differential mRNA expression, in silico analysis of a transcription factor (TF) network correlating with SOCS-1-dependent mRNA expression was carried out and combined with a weighted correlation network analysis (WGCNA)., Results: mRNA analysis in monocyte subsets revealed 46 differentially regulated genes by 2-fold or more. Principal component analysis illustrated a distinct separation of mRNA profiles in monocyte subsets from SOCS-1-deficient mice. Notably, two cell surface receptors crucially involved in the determination of monocyte differentiation and survival, C-X3-C chemokine receptor 1 (CX3CR1) and colony stimulating factor 1 receptor (CSF1R), were identified to be regulated by SOCS-1. Moreover, in silico analysis of a TF network in combination with the WGCNA revealed genes coding for PPAR-γ, NUR77 and several ETSdomain proteins that act as pivotal inflammatory regulators., Conclusion: Our study reveals that SOCS-1 is implicated in a TF network regulating the expression of central transcription factors like PPAR-γ and NUR77 thereby influencing the expression of CX3CR1 and CSF1R that are known to be pivotal for the survival of Ly6C low monocytes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Monocytosis is considered a poor prognostic factor for many cancers, including B cell lymphomas. The mechanisms by which different monocyte subsets support the growth of lymphoma is poorly understood. Using a pre-clinical mouse model of B cell non-Hodgkin's lymphoma (B-NHL), we investigated the impact of tumor progression on circulating monocyte levels, subset distribution and their activity, with a focus on immune suppression. B-NHL development corresponded with significant expansion initially of classical (Ly6Chi) and non-classical (Ly6Clo) monocytes, with accumulation and eventual predominance of Ly6Clo cells. The lymphoma environment promoted the conversion, preferential survival and immune suppressive activity of Ly6Clo monocytes. Ly6Clo monocytes expressed higher levels of immunosuppressive genes including PD-L1/2, Arg1, IDO1 and CD163, compared to Ly6Chi monocytes. Both monocyte subsets suppressed CD8 T cell proliferation and IFN-γ productionin vitro, but via different mechanisms. Ly6Chi monocyte suppression was contact dependent, while Ly6Clo monocytes suppressed via soluble mediators, including IDO and arginase. Ly6Clo monocytes could be selectively depleted in tumor-bearing hosts by liposomal doxorubicin treatment, further enhanced by co-administration of anti-4-1BB monoclonal antibody. This treatment led to a reduction in tumor growth, but failed to improve overall survival. Analogous immunosuppressive monocytes were observed in peripheral blood of diffuse large B cell lymphoma patients and actively suppressed human CD8 T cell proliferation. This study highlights a potential immune evasion strategy deployed by B cell lymphoma involving accumulation of circulating non-classical monocytes with immunosuppressive activity. [ABSTRACT FROM AUTHOR]

A relatively high affinity/avidity of T cell receptor (TCR) recognition for self-peptide bound to major histocompatibility complex II (self-pMHC) ligands is a distinctive feature of CD4 T regulatory (Treg) cells, including their development in the thymus and maintenance of their suppressive functions in the periphery. Despite such high self-reactivity, however, all thymic-derived peripheral Treg populations are neither homogenous in their phenotype nor uniformly immune-suppressive in their function under steady state condition. We show here that based on the previously defined heterogeneity in the phenotype of peripheral Treg populations, Ly6C expression on Treg marks a lower degree of activation, proliferation, and differentiation status as well as functional incompetence. We also demonstrate that Ly6C expression on Treg in a steady state is either up- or downregulated depending on relative amounts of tonic TCR signals derived from its contacts with self-ligands. Interestingly, peripheral appearance and maintenance of these Ly6C-expressing Treg cells largely differed in an age-dependent manner, with their proportion being continuously increased from perinatal to young adult period but then being gradually declined with age. The reduction of Ly6C + Treg in the aged mice was not due to their augmented cell death but rather resulted from downregulation of Ly6C expression. The Ly6C downregulation was accompanied by proliferation of Ly6C + Treg cells and subsequent change into Ly6C - effector Treg with concomitant restoration of immune-suppressive activity. Importantly, we found that this phenotypic and functional change of Ly6C + Treg is largely driven by conventional effector T cell population. Collectively, these findings suggest a potential cross-talk between peripheral Treg subsets and effector T cells and provides better understanding for Treg homeostasis and function on maintaining self-tolerance.

Monocytosis is considered a poor prognostic factor for many cancers, including B cell lymphomas. The mechanisms by which different monocyte subsets support the growth of lymphoma is poorly understood. Using a pre-clinical mouse model of B cell non-Hodgkin's lymphoma (B-NHL), we investigated the impact of tumor progression on circulating monocyte levels, subset distribution and their activity, with a focus on immune suppression. B-NHL development corresponded with significant expansion initially of classical (Ly6Chi) and non-classical (Ly6Clo) monocytes, with accumulation and eventual predominance of Ly6Clo cells. The lymphoma environment promoted the conversion, preferential survival and immune suppressive activity of Ly6Clo monocytes. Ly6Clo monocytes expressed higher levels of immunosuppressive genes including PD-L1/2, Arg1, IDO1 and CD163, compared to Ly6Chi monocytes. Both monocyte subsets suppressed CD8 T cell proliferation and IFN-γ production in vitro , but via different mechanisms. Ly6Chi monocyte suppression was contact dependent, while Ly6Clo monocytes suppressed via soluble mediators, including IDO and arginase. Ly6Clo monocytes could be selectively depleted in tumor-bearing hosts by liposomal doxorubicin treatment, further enhanced by co-administration of anti-4-1BB monoclonal antibody. This treatment led to a reduction in tumor growth, but failed to improve overall survival. Analogous immunosuppressive monocytes were observed in peripheral blood of diffuse large B cell lymphoma patients and actively suppressed human CD8 T cell proliferation. This study highlights a potential immune evasion strategy deployed by B cell lymphoma involving accumulation of circulating non-classical monocytes with immunosuppressive activity.

A series of antibodies against vascular endothelial growth factor (VEGF) have been developed for the treatment of various types of cancer, including non-small cell lung cancer (NSCLC) in recent years. However, tumors frequently demonstrate resistance to these strategies of VEGF inhibition. Efforts to better understand the mechanism underlying the acquired resistance to anti-VEGF antibodies are warranted. In the present study, in order to develop a xenograft model of acquired resistance to anti-VEGF antibody, xenografts of human adenocarcinoma A549 cells were generated through the successive inoculation of tumor tissue explants into first (F1), second (F2) and third (F3) generations of mice treated with the anti-VEGF antibody B20. Tumor growth rate and vessel-forming ability, assessed via cluster of differentiation (CD) 31 staining, were significantly lower in the F1, F2 and F3 groups compared with in the F0 control group (P<0.01), suggesting that drug resistance was not successfully acquired. The percentages of CD11b + myeloid-derived suppressor cells and lymphocyte antigen 6C (Ly6C) + subsets were significantly smaller in F1, F2 and F3 groups compared with in F0 (P<0.01). However, the ratio of Ly6C + to CD11b + cells was significantly higher in the F3 group compared with in F0 and F1 groups (P<0.01), indicating increasing recruitment of the Ly6C + subset with successive challenges with the anti-VEGF antibody. In conclusion, the recruitment of CD11b + Ly6C + monocytes increased with successive generations of NSCLC-xenografted mice challenged by B20, an anti-VEGF agent.

Monocytes are central to the physiopathology of arthritis, but their roles in progression and resolution of the disease remain to be clarified. Using NR4A1 -/- mice, which lack patrolling lymphocyte antigen 6C (Ly6C low ) monocytes, we found that inflammatory Ly6C high monocytes contribute to rapid development of arthritis in a serum transfer-induced arthritis (STIA) model. Our experiments suggest that patrolling monocytes do not promote the initiation and progression of arthritis in mice, as severity of symptoms was amplified in NR4A1 -/- mice. Moreover, we show that treatment of arthritic wild type (WT) mice with cytosporone B (Csn-B), a NR4A1-specific agonist, significantly reduces severity of disease. Effects of Csn-B were absent in monocyte-depleted mice treated with clodronate until Ly6C low monocytes were restored. Adoptive transfer of Ly6C low monocytes in arthritic NR4A1 -/- mice treated with Csn-B reduces joint inflammation, supporting the regulatory role of Ly6C low subset on disease development. Our results also reveal that administration of Csn-B to arthritic mice enhances levels of circulating CD4 + CD25 + FoxP3 + Treg cells, a process requiring the presence of Ly6C low monocytes. Together, these data indicate that Ly6C high monocytes are involved in the initiation and progression of arthritis and Ly6C low monocytes contribute to reduce joint inflammation through the mobilization of Treg cells., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: A key feature of the inflammatory response after cerebral ischemia is the brain infiltration of blood monocytes. There are two main monocyte subsets in the mouse blood: CCR2 + Ly6C hi "inflammatory" monocytes involved in acute inflammation, and CX3CR1 + Ly6C lo "patrolling" monocytes, which may play a role in repair processes. We hypothesized that CCR2 + Ly6C hi inflammatory monocytes are recruited in the early phase after ischemia and transdifferentiate into CX3CR1 + Ly6C lo "repair" macrophages in the brain., Methods: CX3CR1 GFP/+ CCR2 RFP/+ bone marrow (BM) chimeric mice underwent transient middle cerebral artery occlusion (MCAo). Mice were sacrificed from 1 to 28 days later to phenotype and map subsets of infiltrating monocytes/macrophages (Mo/MΦ) in the brain over time. Flow cytometry analysis 3 and 14 days after MCAo in CCR2 -/- mice, which exhibit deficient monocyte recruitment after inflammation, and NR4A1 -/- BM chimeric mice, which lack circulating CX3CR1 + Ly6C lo monocytes, was also performed., Results: Brain mapping of CX3CR1 GFP/+ and CCR2 RFP/+ cells 3 days after MCAo showed absence of CX3CR1 GFP/+ Mo/MΦ but accumulation of CCR2 RFP/+ Mo/MΦ throughout the ischemic territory. On the other hand, CX3CR1 + cells accumulated 14 days after MCAo at the border of the infarct core where CCR2 RFP/+ accrued. Whereas the amoeboid morphology of CCR2 RFP/+ Mo/MΦ remained unchanged over time, CX3CR1 GFP/+ cells exhibited three distinct phenotypes: amoeboid cells with retracted processes, ramified cells, and perivascular elongated cells. CX3CR1 GFP/+ cells were positive for the Mo/MΦ marker Iba1 and phenotypically distinct from endothelial cells, smooth muscle cells, pericytes, neurons, astrocytes, or oligodendrocytes. Because accumulation of CX3CR1 + Ly6C lo Mo/MΦ was absent in the brains of CCR2 deficient mice, which exhibit deficiency in CCR2 + Ly6C hi Mo/MΦ recruitment, but not in NR4A1 -/- chimeric mice, which lack of circulating CX3CR1 + Ly6C lo monocytes, our data suggest a local transition of CCR2 + Ly6C hi Mo/MΦ into CX3CR1 + Ly6C lo Mo/MΦ phenotype., Conclusions: CX3CR1 + Ly6C lo arise in the brain parenchyma from CCR2 + Ly6C hi Mo/MΦ rather than being de novo recruited from the blood. These findings provide new insights into the trafficking and phenotypic diversity of monocyte subtypes in the post-ischemic brain.

The murine Ly6 complex was identified 35 years ago using antisera to lymphocytes. With advances in mAb development, molecular cloning, and genome sequencing, >20 structurally related genes have been identified within this complex on chromosome 15. All members of the Ly6 family and their human homologues share the highly conserved LU domain and most also possess a GPI anchor. Interestingly, many Ly6 proteins are expressed in a lineage-specific fashion, and their expression often correlates with stages of differentiation. As a result, Ly6 proteins are frequently used as surface markers for leukocyte subset identification and targets for antibody-mediated depletion. Murine neutrophils display prominent surface expression of several Ly6 proteins, including Ly6B, Ly6C, and Ly6G. Although the physiology of most Ly6 proteins is not well understood, a role in neutrophil functions, such as migration, is recognized increasingly. In this review, we will provide an overview of the Ly6 complex and discuss, in detail, the specific Ly6 proteins implicated in neutrophil biology.