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

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

Endosomal Sequestration of TLR4 Antibody Induces Myeloid-Derived Suppressor Cells and Reverses Acute Type 1 Diabetes.

We previously showed that treating NOD mice with an agonistic monoclonal anti-TLR4/MD2 antibody (TLR4-Ab) reversed acute type 1 diabetes (T1D). Here, we show that TLR4-Ab reverses T1D by induction of myeloid-derived suppressor cells (MDSCs). Unbiased gene expression analysis after TLR4-Ab treatment demonstrated upregulation of genes associated with CD11b+Ly6G+ myeloid cells and downregulation of T-cell genes. Further RNA sequencing of purified, TLR4-Ab-treated CD11b+ cells showed significant upregulation of genes associated with bone marrow-derived CD11b+ cells and innate immune system genes. TLR4-Ab significantly increased percentages and numbers of CD11b+ cells. TLR4-Ab-induced CD11b+ cells, derived ex vivo from TLR4-Ab-treated mice, suppress T cells, and TLR4-Ab-conditioned bone marrow cells suppress acute T1D when transferred into acutely diabetic mice. Thus, the TLR4-Ab-induced CD11b+ cells, by the currently accepted definition, are MDSCs able to reverse T1D. To understand the TLR4-Ab mechanism, we compared TLR4-Ab with TLR4 agonist lipopolysaccharide (LPS), which cannot reverse T1D. TLR4-Ab remains sequestered at least 48 times longer than LPS within early endosomes, alters TLR4 signaling, and downregulates inflammatory genes and proteins, including nuclear factor-κB. TLR4-Ab in the endosome, therefore, induces a sustained, attenuated inflammatory response, providing an ideal "second signal" for the activation/maturation of MDSCs that can reverse acute T1D.

Analysis of human glioma-associated co-inhibitory immune checkpoints in glioma microenvironment and peripheral blood.

One biomarker for a better therapeutic effect of immune checkpoint inhibitors is high expression of checkpoint in tumor microenvironment The purpose of this study is to investigate the expression of immune checkpoints in human glioma microenvironment and peripheral blood mononuclear cells. First, single-cell suspension from 20 fresh high-grade glioma (HGG) specimens were obtained, and analyzed for lymphocyte composition, then six co-inhibitory immune checkpoints were analyzed at the same time. Second, 36 PBMC specimens isolated from HGG blood samples were analyzed for the same items. In GME, there were four distinct subtypes of cells, among them, immune cells accounted for an average of 51.3%. The myeloid cell population (CD11b+) was the most common immune cell identified, accounting for 36.14% on average; the remaining were most CD3+CD4+ and CD3+/CD8-/CD4- T lymphocytes. In these cells, we detected the expression of BTLA, LAG3, Tim-3, CTLA-4, and VISTA on varying degrees. While in PBMCs, the result showed that when compared with healthy volunteers, the proportion of NK cells decreased significantly in HGG samples (p < 0.01). Moreover, the expression of BTLA, LAG3, and Tim-3 in CD45+ immune cells in PBMC was more remarkable in glioma samples. In conclusion, the CD11b+ myeloid cells were the predominant immune cells in GME. Moreover, some immune checkpoints displayed a more remarkable expression on the immune cells in GME. And the profile of checkpoint expression in PBMC was partially consistent with that in GME.

Graft-Versus-Host Disease Prevention by In Vitro-Generated Myeloid-Derived Suppressor Cells Is Exclusively Mediated by the CD11b+CD11c+ MDSC Subpopulation.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid progenitor cells that dampen overwhelming adaptive immune responses through multiple mechanisms and are recognized as an attractive novel immune intervention therapy for counteracting the destructive effects of graft- versus -host disease (GVHD) developing after allogeneic bone marrow transplantation (BMT). MDSCs can be produced in great numbers for cellular therapy, but they present a mixture of subsets whose functions in GVHD prevention are undefined. Here, we generated MDSCs in vitro from murine BM cells in the presence of GM-CSF and defined the integrin CD11c as a marker to subdivide MDSCs into two functional subgroups: CD11b+CD11c+ and CD11b+CD11c- MDSCs. Isolated CD11b+CD11c+ and CD11b+CD11c- MDSCs both inhibited alloantigen-stimulated T-cell proliferation in vitro, although CD11b+CD11c+ MDSCs were more efficient and expressed higher levels of different immunosuppressive molecules. Likewise, expression of surface markers such as MHC class II, CD80, CD86, or PD-L1 further delineated both subsets. Most importantly, only the adoptive transfer of CD11b+CD11c+ MDSCs into a single MHC class I-disparate allogeneic BMT model prevented GVHD development and strongly decreased disease-induced mortality, while CD11b+CD11c- MDSCs were totally ineffective. Surprisingly, allogeneic T-cell homing and expansion in lymphatic and GVHD target organs were not affected by cotransplanted CD11b+CD11c+ MDSCs indicating a clear contradiction between in vitro and in vivo functions of MDSCs. However, CD11b+CD11c+ MDSCs shifted immune responses towards type 2 immunity reflected by increased Th2-specific cytokine expression of allogeneic T cells. Induction of type 2 immunity was mandatory for GVHD prevention, since CD11b+CD11c+ MDSCs were ineffective if recipients were reconstituted with STAT6-deficient T cells unable to differentiate into Th2 cells. Most importantly, the beneficial graft- versus -tumor (GVT) effect was maintained in the presence of CD11b+CD11c+ MDSCs since syngeneic tumor cells were efficiently eradicated. Strong differences in the transcriptomic landscape of both subpopulations underlined their functional differences. Defining CD11b+CD11c+ MDSCs as the subset of in vitro-generated MDSCs able to inhibit GVHD development might help to increase efficiency of MDSC therapy and to further delineate relevant target molecules and signaling pathways responsible for GVHD prevention.

Cell death and pathological findings of the spleen in COVID-19 patients.

The coronavirus disease 2019(COVID-19) is recognized as systemic inflammatory response syndrome. It was demonstrated that a rapid increase of cytokines in the serum of COVID-19 patients is associated with the severity of disease. However, the mechanisms of the cytokine release are not clear. By using immunofluorescence staining we found that the number of CD11b positive immune cells including macrophages in the spleens of died COVID-19 patients, was significantly higher than that of the control patients. The incidence of apoptosis as measured by two apoptotic markers, TUNEL and cleaved caspase-3, in COVID-19 patients' spleen cells is higher than that in control patients. By double immunostaining CD11b or CD68 and SARS-CoV-2 spike protein, it was found that up to 67% of these immune cells were positive for spike protein, suggesting that viral infection might be associated with apoptosis in these cells. Besides, we also stained the autophagy-related molecules (p-Akt、p62 and BCL-2) in spleen tissues, the results showed that the number of positive cells was significantly higher in COVID-19 group. And compared with non-COVID-19 patients, autophagy may be inhibited in COVID-19 patients. Our research suggest that SARS-CoV-2 may result in a higher rate of apoptosis and a lower rate of autophagy of immune cells in the spleen of COVID-19 patients. These discoveries may increase our understanding of the pathogenesis of COVID-19.

White adipose tissue-infiltrated CD11b+ myeloid cells are a source of S100A4, a new potential marker of hepatic damage.

CONTEXT: The endocrine and immunological properties of subcutaneous vs visceral adipose tissue (sWAT and vWAT, respectively) have turned a milestone in the study of metabolic diseases. The cytokine S100A4 is increased in obesity and has a role in adipose tissue dysfunction. However, the cellular source and its potential role in hepatic damage in obesity has not been elucidated. OBJECTIVE: We aim to study the regulation of S100A4 in immune cells present in sWAT and vWAT, as well as its potential role as a circulating marker of hepatic inflammation and steatosis. DESIGN: A cohort of 60 patients with obesity and distinct metabolic status was analyzed. CD11b+ myeloid cells and T cells were isolated from sWAT and vWAT by magnetic-activating cell sorting, and RNA was obtained. S100A4 gene expression was measured, and correlation analysis with clinical data was performed. Liver biopsies were obtained from 20 patients, and S100A4 circulating levels were measured to check the link with hepatic inflammation and steatosis. RESULTS: S100A4 gene expression was strongly upregulated in sWAT- vs vWAT-infiltrated CD11b+ cells, but this modulation was not observed in T cells. S100A4 mRNA levels from sWAT (and not from vWAT) CD11b+ cells positively correlated with glycemia, triglycerides, TNF-α gene expression and proliferation markers. Finally, circulating S100A4 directly correlated with liver steatosis and hepatic inflammatory markers. CONCLUSION: Our data suggest that sWAT-infiltrated CD11b+ cells could be a major source of S100A4 in obesity. Moreover, our correlations identify circulating S100A4 as a potential novel biomarker of hepatic damage and steatosis.

Functionalization of hybrid surface microparticles for in vitro cellular antigen classification.

Hybrid material surfaces on microparticles are emerging as vehicles for many biomedical multiplexing applications. Functionalization of these hybrid surface microparticles to biomolecules presents unique challenges related to optimization of surface chemistries including uniformity, repeatability, and sample sparring. Hybrid interfaces between microlevel surfaces and individual biomolecules will provide different microenvironments impacting the surface functionalization optimization and efficiency. Here, we propose and validate the first demonstration of streptavidin adsorption-based antibody functionalization on unmodified, hybrid surface microparticles for in vitro analysis. We test this analytical technique and fabricate hybrid surface microparticles with a polystyrene core and aluminum oxide semi-coating. Additionally, we optimize the streptavidin-biotin functionalization chemistry in both assay implementation and sample sparring via analytical mass balances for these microparticles and subsequently conjugate anti-human CD11b antibodies. Result confirmation and characterization occurs from ultraviolet protein absorbance and ImageJ processing of fluorescence microscopy images. Additionally, we design and implement the multi-sectional imaging (MSI) approach to support functionalization uniformity on the hybrid surface microparticles. Finally, as a proof-of-concept performance, we validate anti-CD11b antibodies functionalization by visualizing hybrid surface microparticles conjugate to human neutrophils isolated from blood samples collected from potentially septic patients. Our study introduces and defines a category of functionalization for hybrid surface microparticles with the intent of minuscule sample volumes, low cost, and low environmental impact to be used for many cellular or proteomic in vitro multiplexing applications in the future. Graphical abstract.

Combination of Procalcitonin, CRP and CD11b Biomarkers in Early Detection of Neonatal Sepsis.

The study aimed at comparing the diagnostic performances of CRP, PCT and CD11b in neonatal sepsis and evaluating the effectiveness of the sepsis score system when using a combination of various biomarkers. The study was conducted on 90 neonates divided into 3 equal groups; a group with proven sepsis, suspected sepsis and healthy newborns. All were subjected to measurement of CPR by Latex agglutination, serum Procalcitonin by ELISA and CD11b by flow cytometry. On comparing the three biomarkers; PCT (Serum procalcitonin) was associated with the highest (AUC) area under the curve followed by CD11b and CRP recording the smallest value. However, the AUC of the combined sepsis score was much higher than individual biomarkers. Although the sensitivity of individual biomarkers from procalcitonin to CD11b and lastly CRP but the sensitivity and specificity of the sepsis score showed higher values compared to those of individual biomarkers. In conclusion, the study demonstrate that combination of CRP, CD11b and, procalcitonin can enhance diagnostic discriminative power over traditional tests and overcome the drawbacks of each test alone with greater diagnostic accuracy.

Decrease of Non-Classical and Intermediate Monocyte Subsets in Severe Acute SARS-CoV-2 Infection.

In patients with severe SARS-CoV-2 infection, the development of cytokine storm induces extensive lung damage, and monocytes play a role in this pathological process. Non-classical (NC) and intermediate (INT) monocytes are known to be involved during viral and bacterial infections. In this study, 30 patients with different manifestations of acute SARS-CoV-2 infection were investigated with a flow cytometric study of NC, INT, and classical (CL) monocytes. Significantly reduced NC and INT monocytes and a downregulated HLA-DR were found in acute patients with severe SARS-CoV-2 symptoms. Conversely in patients with moderate symptoms NC and INT monocytes and CD11b expression were increased. © 2020 International Society for Advancement of Cytometry.

Detection of Neutrophils in the Sciatic Nerve Following Peripheral Nerve Injury.

Injury to the sciatic nerve leads to degeneration and debris clearance in the area distal to the injury site, a process known as Wallerian degeneration. Immune cell infiltration into the distal sciatic nerve plays a major role in the degenerative process and subsequent regeneration of the injured motor and sensory axons. While macrophages have been implicated as the major phagocytic immune cell participating in Wallerian degeneration, recent work has found that neutrophils, a class of short-lived, fast responding white blood cells, also significantly contribute to the clearance of axonal and myelin debris. Detection of specific myeloid subtypes can be difficult as many cell-surface markers are often expressed on both neutrophils and monocytes/macrophages. Here we describe two methods for detecting neutrophils in the axotomized sciatic nerve of mice using immunohistochemistry and flow cytometry. For immunohistochemistry on fixed frozen tissue sections, myeloperoxidase and DAPI are used to specifically label neutrophils while a combination of Ly6G and CD11b are used to assess the neutrophil population of unfixed sciatic nerves using flow cytometry.

Correlations of Calf Muscle Macrophage Content With Muscle Properties and Walking Performance in Peripheral Artery Disease.

Background Peripheral artery disease (PAD) is a manifestation of atherosclerosis characterized by reduced blood flow to the lower extremities and mobility loss. Preliminary evidence suggests PAD damages skeletal muscle, resulting in muscle impairments that contribute to functional decline. We sought to determine whether PAD is associated with an altered macrophage profile in gastrocnemius muscles and whether muscle macrophage populations are associated with impaired muscle phenotype and walking performance in patients with PAD. Methods and Results Macrophages, satellite cells, and extracellular matrix in gastrocnemius muscles from 25 patients with PAD and 7 patients without PAD were quantified using immunohistochemistry. Among patients with PAD, both the absolute number and percentage of cluster of differentiation (CD) 11b+CD206+ M2-like macrophages positively correlated to satellite cell number (r=0.461 [P=0.023] and r=0.416 [P=0.042], respectively) but not capillary density or extracellular matrix. The number of CD11b+CD206- macrophages negatively correlated to 4-meter walk tests at normal (r=-0.447, P=0.036) and fast pace (r=-0.510, P=0.014). Extracellular matrix occupied more muscle area in PAD compared with non-PAD (8.72±2.19% versus 5.30±1.03%, P<0.001) and positively correlated with capillary density (r=0.656, P<0.001). Conclusions Among people with PAD, higher CD206+ M2-like macrophage abundance was associated with greater satellite cell numbers and muscle fiber size. Lower CD206- macrophage abundance was associated with better walking performance. Further study is needed to determine whether CD206+ macrophages are associated with ongoing reparative processes enabling skeletal muscle adaptation to damage with PAD. Registration URL: https://www.clini​caltr​ials.gov; Unique identifiers: NCT00693940, NCT01408901, NCT0224660.

Cell Therapy With Mesenchymal Stem Cells Induces an Innate Immune Memory Response That Attenuates Experimental Colitis in the Long Term.

BACKGROUND AND AIMS: Mesenchymal stem cells [MSCs] are used in preclinical and clinical studies for treatment of immune-mediated disorders, thanks to their immunomodulatory properties. Cell therapy with MSCs induces multiple effects in the immune system which ultimately lead to increase in the number of immune cells with regulatory phenotype. In this study, we investigated whether the beneficial effects of MSC therapy are maintained in the long term in a clinically relevant mouse model of colitis. METHODS: A single dose of adipose-derived MSCs [aMSCs] was infused into dextran sulphate sodium [DSS]-induced colitic mice during the induction phase of the disease. Following a latency period of 12 weeks, mice were re-challenged with a second 7-day cycle of DSS. RESULTS: DSS-induced colitic mice treated with aMSCs showed significant reduction in their colitic disease activity index during the second DSS challenge when compared with non-aMSC treated DSS-induced colitic mice. Strikingly, the long-term protection induced by aMSC therapy was also observed in Rag-1-/- mice where no adaptive immune memory cell responses take place. Increased percentages of Ly6G+CD11b+ myeloid cells were observed 12 weeks after the first inflammatory challenge in the peritoneal cavity, spleen, and bone marrow of DSS-induced colitic mice that were infused with aMSCs. Interestingly, upon re-challenge with DSS, these animals showed a concomitant increase in the regulatory/inflammatory macrophage ratio in the colon lamina propria. CONCLUSIONS: Our findings demonstrate for the first time that MSC therapy can imprint an innate immune memory-like response in mice which confers sustained protection against acute inflammation in the long term.

STAT3 signaling in myeloid cells promotes pathogenic myelin-specific T cell differentiation and autoimmune demyelination.

Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the central nervous system. Dysregulation of STAT3, a transcription factor pivotal to various cellular processes including Th17 cell differentiation, has been implicated in MS. Here, we report that STAT3 is activated in infiltrating monocytic cells near active MS lesions and that activation of STAT3 in myeloid cells is essential for leukocyte infiltration, neuroinflammation, and demyelination in experimental autoimmune encephalomyelitis (EAE). Genetic disruption of Stat3 in peripheral myeloid lineage cells abrogated EAE, which was associated with decreased antigen-specific T helper cell responses. Myeloid cells from immunized Stat3 mutant mice exhibited impaired antigen-presenting functions and were ineffective in driving encephalitogenic T cell differentiation. Single-cell transcriptome analyses of myeloid lineage cells from preclinical wild-type and mutant mice revealed that loss of myeloid STAT3 signaling disrupted antigen-dependent cross-activation of myeloid cells and T helper cells. This study identifies a previously unrecognized requisite for myeloid cell STAT3 in the activation of myelin-reactive T cells and suggests myeloid STAT3 as a potential therapeutic target for autoimmune demyelinating disease.

The Kidney Contains Ontogenetically Distinct Dendritic Cell and Macrophage Subtypes throughout Development That Differ in Their Inflammatory Properties.

BACKGROUND: Mononuclear phagocytes (MPs), including macrophages, monocytes, and dendritic cells (DCs), are phagocytic cells with important roles in immunity. The developmental origin of kidney DCs has been highly debated because of the large phenotypic overlap between macrophages and DCs in this tissue. METHODS: We used fate mapping, RNA sequencing, flow cytometry, confocal microscopy, and histo-cytometry to assess the origin and phenotypic and functional properties of renal DCs in healthy kidney and of DCs after cisplatin and ischemia reperfusion-induced kidney injury. RESULTS: Adult kidney contains at least four subsets of MPs with prominent Clec9a-expression history indicating a DC origin. We demonstrate that these populations are phenotypically, functionally, and transcriptionally distinct from each other. We also show these kidney MPs exhibit unique age-dependent developmental heterogeneity. Kidneys from newborn mice contain a prominent population of embryonic-derived MHCIInegF4/80hiCD11blow macrophages that express T cell Ig and mucin domain containing 4 (TIM-4) and MER receptor tyrosine kinase (MERTK). These macrophages are replaced within a few weeks after birth by phenotypically similar cells that express MHCII but lack TIM-4 and MERTK. MHCII+F4/80hi cells exhibit prominent Clec9a-expression history in adulthood but not early life, indicating additional age-dependent developmental heterogeneity. In AKI, MHCIInegF4/80hi cells reappear in adult kidneys as a result of MHCII downregulation by resident MHCII+F4/80hi cells, possibly in response to prostaglandin E2 (PGE2). RNA sequencing further suggests MHCII+F4/80hi cells help coordinate the recruitment of inflammatory cells during renal injury. CONCLUSIONS: Distinct developmental programs contribute to renal DC and macrophage populations throughout life, which could have important implications for therapies targeting these cells.

Activation of the IL-4/STAT6 Signaling Pathway Promotes Lung Cancer Progression by Increasing M2 Myeloid Cells.

Emerging evidence shows that signal transducer and activator of transcription 6 (STAT6) plays critical roles in tumor development. We previously found high-level expression of STAT6 in human lung adenocarcinoma and squamous cell carcinoma, specifically in infiltrated immune cells located in the lung interstitium. Nevertheless, the role of STAT6 signaling in lung carcinogenesis and lung cancer proliferation and its underlying mechanisms remain unclear. This study aimed to investigate the role of STAT6 and the interaction between STAT6 and the tumor microenvironment in pulmonary tumorigenesis. We established a murine model of primary lung carcinogenesis in STAT6-deficient (STAT6-/-) and STAT6 wild-type (WT) BALB/c mice using the carcinogen urethane. Two-month-old male mice were intraperitoneally injected with urethane (1 g/kg) dissolved in phosphate buffered saline (PBS). Primary tumors were monitored in vivo by positron emission tomography scanning. At 4, 6, and 9 months after urethane injection, lung tumors were harvested from the STAT6-/- and WT mice for analysis. Small interfering RNA was used to downregulate the expression of STAT6 in tumor cells. Fluorescence activated cell sorting analysis was used to analyze fluorescence-conjugated cell markers. Transwell assays were used in coculturing experiments. STAT6 protein expression was detected by Western blotting, immunohistochemistry, and immunofluorescence. STAT6 mRNA expression was detected by quantitative real time-polymerase chain reaction. Cell Counting Kit-8 and colony formation assays were performed to evaluate cell proliferation. We detected high expression of STAT6 in CD11b+ cells of lung carcinoma. Our results indicate that STAT6 deficiency inhibits carcinogen-induced tumor growth and improves prognosis. STAT6 deficiency also decreased the mobilization and differentiation of CD11b+ cells. STAT6 deficiency in CD11b+ cells but not tumor cells decreased interleukin (IL)-4 secretion and the differentiation of CD11b+ cells into M2 macrophage cells. In conclusion, our findings indicate that IL-4/STAT6 signaling in CD11b+ cells promotes lung cancer progression by triggering an IL-4 positive feedback loop and increasing M2 myeloid cells. STAT6 may be a new therapeutic target for the prevention and treatment of lung cancer.

Shexiang Tongxin Dropping Pill Improves Peripheral Microvascular Blood Flow via Cystathionine-γ-Lyase.

BACKGROUND To explore the protective effects of Shexiang Tongxin Dropping Pill (STP) in improving peripheral microvascular dysfunction in mice and to explore the involved mechanism. MATERIAL AND METHODS A peripheral microvascular dysfunction model was established by combined myocardial infarction (MI) and lipopolysaccharide (LPS) injection in mice. Then, the mice were randomized into a model group (n=10) or an STP group (n=10), which were treated with normal saline and STP, respectively. The cremaster muscle microvascular blood flow velocity and numbers of leukocytes adherent to the venular wall were evaluated before and after drug intervention. We assessed the expression of adhesion molecule CD11b and related transcript factor FOXO1 in leukocytes, cystathionine-γ-lyase (CSE) mRNA expression in the cremaster muscle, and mitochondrial DNA copy numbers. RESULTS Compared with those of control mice, the cremaster microvascular blood flow velocity, cremaster CSE expression, and mitochondrial DNA copy number in mice from the model group were significantly lower and leukocyte adhesion and CD11b and FOXO1 expression were significantly higher. Intervention with STP could significantly increase the cremaster microvascular flow velocity (0.480±0.010 mm/s vs. 0.075±0.005 mm/s), mRNA expression of cremaster CSE, and mitochondrial DNA copy number, but it inhibited leukocyte adhesion and decreased leukocyte CD11b and FOXO1 expression. CONCLUSIONS STP significantly improved peripheral microcirculation, in which increased CSE expression might be the underlying mechanism.

Red blood cell alloimmunization is associated with lower expression of FcγR1 on monocyte subsets in patients with sickle cell disease.

BACKGROUND: Despite the clinical significance of red blood cell (RBC) alloantibodies, there are currently no laboratory tests available to predict which patients may be at risk of antibody formation after transfusion exposure. Given their phagocytic and inflammatory functions, we hypothesized that differences in circulating monocytes may play a role in alloimmunization. STUDY DESIGN AND METHODS: Forty-two adults with sickle cell disease (SCD) were recruited, with data extracted from the electronic medical record and peripheral blood analyzed by flow cytometry for total monocytes, monocyte subsets (CD14 high/CD16 low+ classical monocytes, CD14 high/CD16 high+ intermediate monocytes, and CD14 intermediate/CD16 high+ non-classical/inflammatory monocytes), and FcγR1 (CD64) expression. Thirteen "non-responder" patients (non-alloimmunized patients with documented RBC transfusion at the study institution) were compared to 20 alloimmunized "responder" patients, who had a total of 44 RBC alloantibodies identified. RESULTS: There were no significant differences in the percentages of total monocytes, monocyte subsets, or measured cytokines between non-responders and responders. However, non-responders had higher CD64 expression on classical monocytes (MFI mean 3424 ± standard deviation 1141) compared to responders (MFI mean 2285 ± 1501), p = 0.029, and on intermediate monocytes (MFI mean 3720 ± 1191) compared to responders (MFI mean 2497 ± 1640), p = 0.033. CONCLUSIONS: Monocytes and the inflammatory milieu increasingly are being appreciated to play a role in some complications of SCD. The differences in FcγR1 expression on monocyte subsets noted between responders and non-responders, which cannot be directly explained by the serum cytokines evaluated, warrant further investigation.

Glutamine Antagonist JHU083 Normalizes Aberrant Glutamate Production and Cognitive Deficits in the EcoHIV Murine Model of HIV-Associated Neurocognitive Disorders.

HIV-associated neurocognitive disorders (HAND) have been linked to dysregulation of glutamate metabolism in the central nervous system (CNS) culminating in elevated extracellular glutamate and disrupted glutamatergic neurotransmission. Increased glutamate synthesis via upregulation of glutaminase (GLS) activity in brain immune cells has been identified as one potential source of excess glutamate in HAND. However, direct evidence for this hypothesis in an animal model is lacking, and the viability of GLS as a drug target has not been explored. In this brief report, we demonstrate that GLS inhibition with the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) can reverse cognitive impairment in the EcoHIV-infected mouse model of HAND. However, due to peripheral toxicity DON is not amenable to clinical use in a chronic disease such as HAND. We thus tested JHU083, a novel, brain penetrant DON prodrug predicted to exhibit improved tolerability. Systemic administration of JHU083 reversed cognitive impairment in EcoHIV-infected mice similarly to DON, and simultaneously normalized EcoHIV-induced increases in cerebrospinal fluid (CSF) glutamate and GLS activity in microglia-enriched brain CD11b + cells without observed toxicity. These studies support the mechanistic involvement of elevated microglial GLS activity in HAND pathogenesis, and identify JHU083 as a potential treatment option. Graphical Abstract Please provide Graphical Abstract caption.Glutamine Antagonist JHU083 Normalizes Aberrant Glutamate Production and Cognitive Deficits in the EcoHIV Murine Model of HIV-Associated Neurocognitive Disorders .

Modulation of MS-like disease by a multi epitope protein is mediated by induction of CD11c+CD11b+Gr1+ myeloid-derived dendritic cells.

Specific neutralization of the pathogenic autoimmune cells is the ultimate goal in therapy of Multiple Sclerosis (MS). However, the pathogenic autoimmunity in MS, can be directed against several major target antigens, and therefore targeting pathogenic T-cells directed against a single target antigen is unlikely to be effective. To overcome this multiplicity and the potential complexity of pathogenic autoreactivities in MS, we have put forward the concept of concomitant multi-antigen/multi-epitope targeting as, a conceivably more effective approach to immunotherapy of MS. We constructed an (Experimental Autoimmune Encephalomeylitis (EAE)/MS-related synthetic human Target Autoantigen Gene (MS-shMultiTAG) designed to encode in tandem only EAE/MS related epitopes of all known encephalitogenic proteins. The MS-related protein product (designated Y-MSPc) was immunofunctional and upon tolerogenic administration, it effectively suppressed and reversed EAE induced by a single encephalitogenic protein. Furthermore, Y-MSPc also fully abrogated the development of "complex EAE" induced by a mixture of five encephalitogenic T-cell lines, each specific for a different encephalitogenic epitope of MBP, MOG, PLP, MOBP and OSP. Strikingly, Y-MSPc was consistently more effective than treatment with the single disease-specific peptide or with the peptide cocktail, both in suppressing the development of "classical" or "complex" EAE and in ameliorating ongoing disease. Overall, the modulation of EAE by Y-MSPc was associated with anergizing the pathogenic autoreactive T-cells, downregulation of Th1/Th17 cytokine secretion and upregulation of TGF-ß secretion. Moreover, we show that both suppression and treatment of ongoing EAE by tolerogenic administration of Y-MSPc is associated also with a remarkable increase in a unique subset of dendritic-cells (DCs), CD11c+CD11b+Gr1+-myeloid derived DCs in both spleen and CNS of treated mice. These DCs, which are with strong immunoregulatory characteristics and are functional in down-modulation of MS-like-disease displayed increased production of IL-4, IL-10 and TGF-ß and low IL-12. Functionally, these myeloid DCs suppress the in-vitro proliferation of myelin-specific T-cells and more importantly, the cells were functional in-vivo, as their adoptive transfer into EAE induced mice resulted in strong suppression of the disease, associated with a remarkable induction of CD4 + FoxP3+ regulatory cells. These results, which highlight the efficacy of "multi-epitope-targeting" agent in induction of functional regulatory CD11c+CD11b+Gr1+myeloid DCs, further indicate the potential role of these DCs in maintaining peripheral tolerance and their involvement in downregulation of MS-like-disease.

The transcriptional profile of circulating myeloid derived suppressor cells correlates with tumor development and progression in mouse.

Myeloid derived suppressor cells (MDSCs) play key roles in cancer development. Accumulation of peripheral-blood MDSCs (PB-MDSCs) corresponds to the progression of various cancers, but provides only a crude indicator. We aimed toward identifying changes in the transcriptional profile of PB-MDSCs in response to tumor growth. CT26 colon cancer cells and B16 melanoma cells (106) were inoculated into peritoneal cavities of BALB/c mice and subcutaneously to C57-black mice, respectively. The circulating levels and global transcriptional patterns of PB CD11b+Ly6g+ MDSCs were assessed in control mice, and 4, 8, and 11 days following tumor cell inoculation. Although a significant accumulation of PB-MDSCs was demonstrated only 11 days following tumor induction, a pronounced transcriptional response was identified already on day 4 while the tumor was ~1 mm in size. Further transcriptional changes correlated with different stages of tumor growth. Key MDSC genes and canonical signaling pathways were activated along tumor progression. This phenomenon was demonstrated in both cancer models, and a consensus set of 817 genes, involved in myeloid cell recruitment and angiogenesis, was identified. The data suggest that the transcriptional signatures of PB-MDSC may serve as markers for tumor progression, as well as providing potential targets for future therapies.