LFA-1 Research Articles

Liver Regeneration Following Thermal Ablation Using Nanocarrier Mediated Targeted Mesenchymal Stem Cell Therapy

Abstract Purpose To test the efficacy of nanocarrier (NC) mediated mesenchymal stem cell (MSC) therapy for liver regeneration following thermal ablation of porcine livers. Materials and Methods Liver radiofrequency ablation was performed in 18 swines divided into MSC, MSC + NC and control groups. The test groups received infusion of MSC or MSC + NC labeled with enhanced green fluorescent protein (eGFP) via hepatic artery. MSC + NC group had MSCs coated with dendrimer nanocarrier complexed with I-Domain of lymphocyte function-associated antigen-1 (LFA-1). Nanocarriers direct homing of MSCs by binding to its counterpart protein, intercellular adhesion molecule-1 (ICAM-1), which is overexpressed at the periablation margins from inflammation. Ablation cavity reduction by CT volumetry was used as surrogate marker for liver regeneration. Cell proliferation was assessed with Ki67 and HepPar-1 stains. GFP identified MSC derived cells. Results Total number of ablations in control animals were 13 across 4 animals. In the MSC group, there were 23 ablations across 6 animals, and in MSC + NC group there were 21 ablations across 6 animals. Ablation cavity volume reduction from day 0 to 30 were 64.4 ± 15.0%, 61.5 ± 12.9% and 80.3 ± 9.4% for control, MSC and MSC + NC groups, respectively (MSC + NC vs MSC: p < 0.001, MSC + NC vs. control: p = 0.001). GFP+ cell count at margins was 426.8 ± 193.2 for MSC group and 498.6 ± 235.2 for MSC + NC group (p = 0.01). The mean Ki67 and HepPar-1 staining at margins were 9.81 ± 4.5% and 6.12 ± 4.2% for MSC + NC group versus 7.59 ± 3.7% and 5.09 ± 3.7% for MSC group, respectively (P < 0.001 and P = 0.09, respectively). Conclusion Nanocarrier-mediated MSC therapy promotes liver regeneration by engrafting MSCs at ablation margins, potentially making liver-directed therapy viable for patients with severe liver dysfunction. This technology may also benefit other solid organs. Graphical Abstract

Targeting ICAM1 to Ameliorate Vaso-Occlusion and Inflammation in Sickle Cell Disease.

Sickle cell disease (SCD) is a hereditary disorder characterized by vaso-occlusion, inflammation, and tissue damage. Intercellular adhesion molecule 1 (ICAM-1) plays a crucial role in the pathophysiology of SCD by promoting the adhesion of sickle cells to the endothelium, contributing to vaso-occlusion and tissue damage. The ICAM-1 gene encodes a glycoprotein that interacts with lymphocyte function-associated antigen 1 (LFA-1) and macrophage 1-antigen (Mac-1) receptors, perpetuating inflammation, and oxidative stress. The NF-κB signaling pathway regulates ICAM-1 expression, which is elevated in patients with SCD, leading to increased endothelial cell activation and damage. Targeting ICAM-1 and its interactions with sickle cells and the endothelium has emerged as a potential therapeutic strategy for managing SCD. This review highlights the complex interplay between ICAM-1, sickle cells, and the endothelium, and discusses the potential of ICAM-1-targeted therapies for mitigating VOC and improving the quality of life for patients with SCD.

Inhibition of Mac-1 allows human macrophages to migrate against the direction of shear flow on ICAM-1.

All immune cells must transit from the blood to distal sites such as the lymph nodes, bone marrow, or sites of infection. Blood borne monocytes traffic to the site of inflammation by adhering to the endothelial surface and migrating along endothelial intracellular adhesion molecule 1 (ICAM-1) by their ligand's macrophage 1 antigen (Mac-1) and lymphocyte functional antigen 1 (LFA-1) to transmigrate through the endothelium. Poor patient prognoses in chronic inflammation and tumors have been attributed to the hyper recruitment of certain types of macrophages. Therefore, targeting the binding of ICAM-1 to its respective ligands provides a novel approach to targeting the recruitment of macrophages. To that end, we determined whether the loss of Mac-1 expression could induce this upstream migration behavior by using blocking antibodies against Mac-1 to examine the effects of hydrodynamic flow on the migration of the human macrophage cell line U-937 on ICAM-1 surfaces. Blocking Mac-1 on U-937 cells led to upstream migration against the direction of shear flow on ICAM-1 surfaces. In sum, the ability of macrophages to migrate upstream when Mac-1 is blocked represents a new avenue to precisely control the differentiation, migration, and trafficking of macrophages.

A Multifunctional Biomimetic Nanoplatform for Dual Tumor Targeting-Assisted Multimodal Therapy of Colon Cancer.

The biomimetic nanoparticles (NPs) possessing abilities of tumor targeting and multimodal therapy show great potential for efficient combat of colon cancer. Herein, we developed a multifunctional biomimetic nanoplatform (Fe3O4@PDA@CaCO3-ICG@CM) based on CaCO3-modified magnetic polydopamine (PDA) loaded with indocyanine green (ICG), which was encapsulated by a mouse lymphoma cell (EL4) membrane (CM) expressing functional proteins (i.e., lymphocyte function-associated antigen 1, LFA-1; transforming growth factor-β receptor, TGF-βR; programmed cell death protein 1, PD-1; and factor related apoptosis ligand, FasL). Under magnetic attraction and LFA-1/PD-1-mediated endocytosis, Fe3O4@PDA@CaCO3-ICG@CM efficiently targeted CT26 colon tumor cells. The released calcium ion (Ca2+) from the NPs triggered by acidic tumor microenvironment, the enhanced photothermal effect contributed by the combination of PDA and ICG, and FasL's direct killing effect together induced tumor cells apoptosis. Moreover, the apoptosis of CT26 cells induced immunogenic cell death (ICD) to promote the maturation of dendritic cells (DCs) to activate CD4+/CD8+ T cells, thereby fighting against tumor cells, which could further be boosted by programmed death-ligand 1 (PD-L1) blockage and transforming growth factor-β (TGF-β) scavenging by Fe3O4@PDA@CaCO3-ICG@CM. As a result, in vivo satisfactory therapeutic effect was observed for CT26 tumor bearing-mice treated with Fe3O4@PDA@CaCO3-ICG@CM under laser irradiation and magnetic attraction, which could eradicate primary tumors and restrain distant tumors through dual tumor targeting-assisted multimodal therapy and eliciting adaptive antitumor immune response, generating the immune memory for inhibiting tumor metastasis and recurrence. Taken together, the multifunctional biomimetic nanoplatform exhibits superior antitumor effects, providing an insightful strategy for the field of nanomaterial-based treatment of cancer.

Mesenchymal Stem Cell-Based Biomimetic Liposome for Targeted Treatment of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disorder that severely compromises joint health. The primary therapeutic strategy for advanced RA aims to inhibit joint inflammation. However, the nonspecific distribution of pharmacological agents has limited therapeutic efficacy and heightens the risks associated with RA treatment. To address this issue, we developed mesenchymal stem cell (MSC)-based biomimetic liposomes, termed MSCsome, which were composed of a fusion between MSC membranes and liposomes. MSC some with relatively simple preparation method effectively enhanced the targeting efficiency of drug to diseased joints. Interaction between lymphocyte function-associated antigen-1 and intercellular adhesion molecule-1 enhanced the affinity of the MSCsome for polarized macrophages, thereby improving its targeting capability to affected joints. The effective targeted delivery facilitated drug accumulation in joints, resulting in the significant inhibition of the inflammation, as well as protection and repair of the cartilage. In conclusion, this study introduced MSCsome as a promising approach for the effective treatment of advanced RA, providing a novel perspective on targeted drug delivery therapy for inflammatory diseases.

Low-affinity LFA1-dependent outside-in signaling mediates avidity modulation via the Rabin8-Rab8 axis.

Lymphocyte interactions mediated by leukocyte integrin lymphocyte function-associated antigen 1 (LFA1) and intercellular adhesion molecules (ICAMs) are important for lymphocyte trafficking and antigen recognition. Integrins are regulated by the modulation of ligand-binding affinity and avidity (valency). Although the mechanism underlying high-affinity LFA1 binding has been investigated extensively, the molecular mechanisms by which low-affinity multivalent binding initiates adhesion remain unclear. We previously showed that ICAM1 and monoclonal antibodies that recognize specific LFA1 conformations induce the accumulation of LFA1 at the contact surface. In this study, we found that the small GTPase Rab8 is critical for intracellular transport and accumulation of LFA1 at cell contact areas mediated by low-affinity LFA1-dependent outside-in signaling. Super-resolution microscopy revealed that Rab8 co-localized with LFA1 in small vesicles near the contact membrane. Inactivation of Rab8 decreased ICAM1-dependent adhesion and substantially reduced LFA1 density on the contact membrane. The GTP-bound active form of Rab8 increased cell adhesiveness and promoted LFA1 accumulation at the contact area through co-trafficking with LFA1. Rab8 activation was induced by low-affinity conformation-dependent outside-in signaling via the guanine exchange factor Rabin8, which induced Rab8 activation at the cell contact area independent of Rap1. Single-molecule imaging of ICAM1 on a supported planner lipid bilayer demonstrated that Rab8 increased the frequency of LFA1-ICAM1 interactions without affecting their binding lifetime, indicating that Rab8 is mainly involved in the modulation of LFA1 avidity rather than LFA1 affinity. The present findings underscore the importance of low-affinity conformation-dependent outside-in signaling via the Rabin8-Rab8 axis leading to the initiation of LFA1 transport to the contact area.

Viral modulation of type II interferon increases T cell adhesion and virus spread

During primary varicella zoster virus (VZV) infection, infected lymphocytes drive primary viremia, causing systemic dissemination throughout the host, including the skin. This results in cytokine expression, including interferons (IFNs), which partly limit infection. VZV also spreads from skin keratinocytes to lymphocytes prior to secondary viremia. It is not clear how VZV achieves this while evading the cytokine response. Here, we show that VZV glycoprotein C (gC) binds IFN-γ and modifies its activity, increasing the expression of a subset of IFN-stimulated genes (ISGs), including intercellular adhesion molecule 1 (ICAM1), chemokines and immunomodulatory genes. The higher ICAM1 protein level at the plasma membrane of keratinocytes facilitates lymphocyte function-associated antigen 1-dependent T cell adhesion and expression of gC during infection increases VZV spread to peripheral blood mononuclear cells. This constitutes the discovery of a strategy to modulate IFN-γ activity, upregulating a subset of ISGs, promoting enhanced lymphocyte adhesion and virus spread.

Abstract PO-035: Genome-wide CRISPR knockout screen identifies sialylation as a tumor suppressive mechanism in B-cell malignancies

Abstract Ba/F3 is a murine pro-B cell line that is dependent on interleukin-3 (IL-3) for its survival and proliferation. It has been widely used for identifying oncogenes and mechanisms of drug-resistance. Using this cell line, we conducted a genome-wide CRISPR knockout screen to identify genes whose loss can confer IL-3 independence, hence may be implied as tumor suppressor genes (TSGs). We identified several genes that map to human chromosome 6q, a frequent deletion hotspot in B-cell malignancies, that may act as TSGs. One of these putative novel TSGs is Slc35a1, which encodes the membrane solute carrier that facilitates the transport of nucleotide sugar (CMP-sialic acid) into the Golgi apparatus for subsequent sialylation. Interestingly, in addition to Slc35a1, pathway analysis of hits from the CRISPR screen identified several genes in the sialic acid metabolism pathway, Nans, Cmas, and St3gal5. We confirmed that their silencing led to decreased protein sialyation and could confer IL-3-independent cell survival and growth in Ba/F3 cells. We continued to characterize Slc35a1 since its homozygous and heterozygous deletions are seen in 7-40% of different types of B-cell malignancies, including approximately 40% of patients with Diffuse Large B-cell Lymphoma. Slc35a1 deletion in murine and human cell-lines conferred cytokine-independent growth. Tail-vein injection of Slc35a1-silenced Ba/F3 cells resulted in allograft tumor growth presenting with splenomegaly and lymphadenopathy. Using Maackia amurensis lectin II (MAL II) to pull down sialylated proteins for mass spectrometry, we identified proteins that were differentially sialylated in Slc35a1 wild-type and knockout cells, among which are the α and β subunits of the integrin receptor, lymphocyte functional antigen 1 (LFA-1). As integrin desialylation can promote its constitutive activation, we are currently studying the impact of LFA-1 sialylation on B-cell function and transformation. We have also developed mouse models with B-cell specific ablation of Nans and Slc35a1 to further evaluate the role of sialylation on B-cell development and tumorigenesis in vivo. Overall, our study demonstrates that sialylation may function as a tumor suppressive mechanism and serve as a therapeutic target in B-cell cancers. Citation Format: Namratha Sheshadri, Rongrong Li, Muhammad Usama Tariq, Jianliang Shen, Jaeyong Jung, Junrong Yan, Kevin Lu, Zhiyuan Shen, Ping Xie, Wei-Xing Zong. Genome-wide CRISPR knockout screen identifies sialylation as a tumor suppressive mechanism in B-cell malignancies [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-035.

Red wine alleviates atherosclerosis-related inflammatory markers in healthy subjects rather than in high cardiovascular risk subjects: A systematic review and meta-analysis.

Moderate red wine (RW) consumption is associated with a low risk of cardiovascular disease (CVD). However, few studies have evaluated the effects of RW and white wine (WW) on inflammatory markers related to atherosclerosis in healthy individuals and high-risk subjects for CVD. This study aimed to assess the effect of RW on inflammatory markers in healthy individuals and high-risk subjects for CVD compared with moderate alcohol consumption. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses 2020 (PRISMA) was followed in this study. The PubMed, Embase, Cochrane, Web of Science, SinoMed, EbscoHost, and ScienceDirect databases were searched. The risk of bias and quality of the included trials were assessed using the Cochrane Handbook. The main results are summarized in Stata 12. Twelve studies were included in the meta-analysis. The results demonstrated that RW significantly decreased circulating intercellular cell adhesion molecule-1, vascular cell adhesion molecule-1 (VCAM-1), tumor necrosis factor-alpha (TNF-α), lymphocyte function-associated antigen-1, and Sialyl-Lewis X expression on the surface of monocytes in healthy subjects, but not in patients with CVD. Additionally, RW significantly decreased Sialyl-Lewis X but increased clusters of differentiation 40 (CD40) expressed on the surface of T lymphocytes and significantly decreased C-C chemokine receptor type 2 (CCR2) and very late activation antigen 4 (VLA-4) expressed on the surface of monocytes. Interestingly, subgroup analysis also found that RW significantly decreased circulating interleukin-6 (IL-6) in Spain but not in other countries, and significantly increased αMβ2 (Mac-1) in the group that had an intervention duration of less than 3 weeks. Moderate consumption of RW is more effective than WW in alleviating atherosclerosis-related inflammatory markers in healthy people rather than high-risk subjects for CVD, but this needs to be further confirmed by studies with larger sample sizes.

Therapeutic Applications of Aggregatibacter actinomycetemcomitans Leukotoxin.

Aggregatibacter actinomycetemcomitans is a Gram-negative oral bacterium that has been primarily studied for its role in causing periodontal disease. The bacterium has also been implicated in several systemic diseases such as endocarditis and soft tissue abscesses. Leukotoxin (LtxA) is perhaps the best studied protein virulence factor from A. actinomycetemcomitans. The protein can rapidly destroy white blood cells (WBCs), helping the bacterium to subvert the host immune system. The functional receptor for LtxA is lymphocyte function associated antigen-1 (LFA-1), which is expressed exclusively on the surfaces of WBCs. Bacterial expression and secretion of the protein are highly regulated and controlled by a number of genetic and environmental factors. The mechanism of LtxA action on WBCs varies depending on the type of cell that is being killed, and the protein has been shown to activate numerous cell death pathways in susceptible cells. In addition to serving as an important virulence factor for the bacterium, because of its exquisite specificity and rapid activity, LtxA is also being investigated as a therapeutic agent that may be used to treat diseases such as hematological malignancies and autoimmune/inflammatory diseases. It is our hope that this review will inspire an increased intensity of research related to LtxA and its effect on Aggressive Periodontitis, the disease that led to its initial discovery.

A specific chiral HPLC method for lifitegrast and determination of enantiomeric impurity in drug substance, ophthalmic product and stressed samples

Lifitegrast is a lymphocyte function-associated antigen-1 (LFA-1) antagonist used to treat the indications and symptoms associated with dry eye disease (DED), one of the most common ocular surface diseases. Lifitegrast has a chiral center, and the S-enantiomer (S-Lif) is responsible for the therapeutic effects, while the R-enantiomer (R-Lif) lacks efficacy in the treatment of DED. Lifitegrast ophthalmic solution containing 5% lifitegrast was approved by the United States Food and Drug Administration (FDA) in July 2016 for the treatment of DED in patients 17 years of age and older. The objective of this study was to develop a chiral HPLC method for the determination of the enantiomeric impurity of lifitegrast in the drug substance and in the ophthalmic product. In addition, we aimed to investigate the effect of stress and stability conditions on the enantiomeric purity of lifitegrast in both drug substance and ophthalmic solution. During the method development studies, four known lifitegrast impurities (Lif. Imp. A-D) and stressed lifitegrast samples were injected to ensure the specificity of the developed method. The enantiomers of lifitegrast are well separated with a resolution of higher than 4.0. They are also well separated from the peaks of the diluent, impurities, and the placebo used to prepare the ophthalmic solution without interference in 20 min. Chiral separation was achieved using a Chiralpak AD-H column (250 × 4.6 mm, 5.0 μm) at 40 °C with a mobile phase consisting of a mixture of n-hexane, 2-propanol, and formic acid (500:500:2, v/v/v) at a flow rate of 1.0 mL/min and a detection wavelength of 260 nm. Methanol was used as the diluent, and the drug substance solution was found to be stable for 48 h at 15 °C. The optimized chiral HPLC method for lifitegrast was validated according to ICH Q2, and the calibration curves showed excellent linearity for R-Lif (0.0369 – 1.816 µg/mL). This is the first stability-indicating, specific / selective, sensitive, linear, precise, accurate, and robust chiral HPLC method for the determination of R-Lif in S-Lif. The amount of enantiomeric impurity R-Lif in S-Lif increased under all stress and photostability test conditions without exceeding the acceptable impurity limit, with the most significant increase observed at elevated temperatures (105 °C) for both the drug substance in powder form and the ophthalmic drug solution.

Endoplasmic Reticulum Protein 72 Regulates Integrin Mac-1 Activity to Influence Neutrophil Recruitment.

Integrins mediate the adhesion, crawling, and migration of neutrophils during vascular inflammation. Thiol exchange is important in the regulation of integrin functions. ERp72 (endoplasmic reticulum-resident protein 72) is a member of the thiol isomerase family responsible for the catalysis of disulfide rearrangement. However, the role of ERp72 in the regulation of Mac-1 (integrin αMβ2) on neutrophils remains elusive. Intravital microscopy of the cremaster microcirculation was performed to determine in vivo neutrophil movement. Static adhesion, flow chamber, and flow cytometry were used to evaluate in vitro integrin functions. Confocal fluorescent microscopy and coimmunoprecipitation were utilized to characterize the interactions between ERp72 and Mac-1 on neutrophil surface. Cell-impermeable probes and mass spectrometry were used to label reactive thiols and identify target disulfide bonds during redox exchange. Biomembrane force probe was performed to quantitatively measure the binding affinity of Mac-1. A murine model of acute lung injury induced by lipopolysaccharide was utilized to evaluate neutrophil-associated vasculopathy. ERp72-deficient neutrophils exhibited increased rolling but decreased adhesion/crawling on inflamed venules in vivo and defective static adhesion in vitro. The defect was due to defective activation of integrin Mac-1 but not LFA-1 (lymphocyte function-associated antigen-1) using blocking or epitope-specific antibodies. ERp72 interacted with Mac-1 in lipid rafts on neutrophil surface leading to the reduction of the C654-C711 disulfide bond in the αM subunit that is critical for Mac-1 activation. Recombinant ERp72, via its catalytic motifs, increased the binding affinity of Mac-1 with ICAM-1 (intercellular adhesion molecule-1) and rescued the defective adhesion of ERp72-deficient neutrophils both in vitro and in vivo. Deletion of ERp72 in the bone marrow inhibited neutrophil infiltration, ameliorated tissue damage, and increased survival during murine acute lung injury. Extracellular ERp72 regulates integrin Mac-1 activity by catalyzing disulfide rearrangement on the αM subunit and may be a novel target for the treatment of neutrophil-associated vasculopathy.

LFA-1 regulated by IL-2/STAT5 pathway boosts antitumor function of intratumoral CD8+ T cells for improving anti-PD-1 antibody therapy

ABSTRACT Anti-PD-1 antibody therapy has achieved success in tumor treatment; however, the duration of its clinical benefits are typically short. The functional state of intratumoral CD8+ T cells substantially affects the efficacy of anti-PD-1 antibody therapy. Understanding how intratumoral CD8+ T cells change will contribute to the improvement in anti-PD-1 antibody therapy. In this study, we found that tumor growth was not arrested after the late administration of anti-PD-1 antibody and that the antitumor function of CD8+ T cells decreased with tumor progression. The results of the RNA sequencing of CD8+ T cells infiltrating the tumor site on days 7 and 14 showed that the cell adhesion molecule Lymphocyte Function-associated Antigen-1 (LFA-1) participates in regulating the antitumor function of CD8+ T cells and that decreased LFA-1 expression in intratumoral CD8+ T cells is associated with tumor progression. By analyzing the Gene Expression Omnibus (GEO) database and our results, we found that the antitumor function of intratumoral CD8+ T cells with high LFA-1 expression was stronger. The formation of immune synapses is impaired in Itgal-si CD8+ T cells, resulting in decreased anti-tumor function. LFA-1 expression in intratumoral CD8+ T cells is regulated by the IL-2/STAT5 pathway. The combination of IL-2 and anti-PD-1 antibody effectively enhanced LFA-1 expression and the antitumor function of intratumoral CD8+ T cells. The adoptive transfer of OT-1 T cells overexpressing LFA-1, STAT5A, or STAT5B resulted in higher antitumor function, deferred tumor growth, and prolonged survival. These findings indicate that LFA-1-mediated immune synapse acts as a regulator of the antitumor function of intratumoral CD8+ T cells, which can be applied to improve anti-PD-1 antibody therapy.

Isolation and characterization of a novel single-chain variable fragment (scFv) against Lymphocyte function-associated antigen-1 (LFA-1) using phage display method.

Lymphocyte function-associated antigene-1 (LFA-1) is a well-described integrin found on lymphocytes and other leukocytes, which is known to be overexpressed in leukemias and lymphomas. This receptor plays a significant role in immune responses such as T-cell activation, leukocyte cell-cell interactions, and trafficking of leukocyte populations. Subsequently, binders of LFA-1 emerge as potential candidates for cancer and autoimmune therapy. This study used the phage display technique to construct and characterize a high-affinity single-chain fragment variable (scFv) antibody against LFA-1. After expression, purification, dialysis, and concentration of the recombinant LFA-1 protein, four female BALB/c mice were immunized, splenocyte's mRNA was extracted, and cDNA was synthesized. A scFv library was constructed by linking the amplified VH/Vκ fragments through a 72-bp linker using SOEing PCR. Next, the scFv gene fragments were cloned into the pComb-3XSS phagemid vector; thus, the phage library was developed. The selection process involved three rounds of phage-bio-panning, polyclonal, and monoclonal phage ELISA. AF17 was chosen and characterized among the positive clones through SDS-PAGE, Western blotting, indirect ELISA, and in-silico analyses. The results of the study showed the successful construction of a high-affinity scFv library against LFA-1. The accuracy of the AF17 production and its ability to bind to the LFA-1 were confirmed through SDS-PAGE, Western blot, and ELISA. This study highlights the potential application of the high-affinity AF17 against LFA-1 for targeting T lymphocytes for therapeutic purposes.

Erythropoietin Treatment in Mice Enriches Bone Marrow Osteoclast Precursors, As Reflected By Increased Macrophage Colony-Stimulating Factor (MCSF) and Elevated Expression of Osteoclast Markers in Monocytes

The impact of erythropoietin (EPO) on erythropoiesis has long been established. However, recent studies have indicated that EPO may also exert regulatory effects on other hematopoietic lineages, including the monocyte lineage. In that respect, we have previously shown that EPO treatment in mice induces bone loss. Here, we studied the effect of EPO on bone marrow (BM) monocytes and its potential implications for osteoclastogenesis. To address the dynamic changes in monocyte populations following EPO administration, we injected EPO (180U) once, or 3 times on alternating days for one week. Mice were sacrificed 16 h after EPO injection. Our results revealed a significant 20 percent increase in the monocyte population (TER119 -, LY6G -, CD11b +) 16 hours after EPO injection, and a twofold increase in the levels of these cells after one week of 3 injections of EPO. Soluble macrophage colony-stimulating factor (MCSF) protein levels in the bone marrow were increased 1.5-fold 16 h after EPO injection, while MCSF mRNA expression levels remained unchanged. After 1-week of EPO administration (3 injections), although the global BM expression of MCSF was reduced, we observed a two-fold increase in MCSF expression levels in T cells (CD3 +). To further elucidate the functional consequences of EPO-induced monocyte expansion, we isolated monocytes from mice treated with EPO for one week. We characterized expression levels of key osteoclastic markers in these cells. Indeed, we found that in BM monocytes, EPO treatment led to the upregulation of Lymphocyte function-associated antigen 1 (LFA-1) and Receptor Activator of Nuclear Factor κ B (RANK). The former is implicated in osteoclastogenesis via its interaction with intercellular adhesion molecule-1 (ICAM−1), while the latter is the surface receptor for RANK ligand which regulates osteoclast differentiation and activation. These data suggest that EPO not only influences BM monocyte expansion, but also contributes to the regulation of osteoclast differentiation, thus linking erythropoiesis to bone homeostasis. Furthermore, we investigated the early effects of EPO treatment on the expression of transcription factors implicated in monocyte differentiation. Remarkably, we found that EPO administration for 16 hours resulted in the downregulation of KLF2 and KLF4, transcription factors involved in monocytic lineage commitment. The rapid EPO-induced expansion of the monocytic population, coupled with the EPO-regulated expression of MSCF, osteoclastic markers, and transcription factors, suggests a role for EPO in modulating monocyte-to-osteoclast differentiation. The current study opens avenues for further investigating the crosstalk between erythropoiesis, myelopoiesis, and bone homeostasis.

CCL5 promotes LFA-1 expression in Th17 cells and induces LCK and ZAP70 activation in a mouse model of Parkinson's disease.

Parkinson's disease (PD), which is associated to autoimmune disorders, is characterized by the pathological deposition of alpha-synuclein (α-Syn) and loss of dopaminergic (DA) neurons. Th17 cells are thought to be responsible for the direct loss of DA neurons. C-C chemokine ligand 5 (CCL5) specifically induces Th17 cell infiltration into the SN. However, the specific effect of CCL5 on Th17 cells in PD and the relationship between CCL5 and lymphocyte function-associated antigen-1 (LFA-1) expression in Th17 cells are unknown. We evaluated the effects of CCL5 on LFA-1 expression in Th17 cells in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and examined Th17 cell differentiation upon CCL5 stimulation in vitro. Furthermore, we assessed the effects of CCL5 on tyrosine kinase zeta-chain-associated protein kinase 70 (ZAP70) and lymphocyte-specific protein tyrosine kinase (LCK) activity in CCL5-stimulated Th17 cells in vivo and in vitro. CCL5 increased the proportion of peripheral Th17 cells in MPTP-treated mice, LFA-1 expression on Th17 cells, and Th17 cell levels in the SN of MPTP-treated mice. CCL5 promoted Th17 cell differentiation and LFA-1 expression in naive T cells in vitro. Moreover, CCL5 increased Th17 cell differentiation and LFA-1 expression by stimulating LCK and ZAP70 activation in naive CD4+ T cells. Inhibiting LCK and ZAP70 activation reduced the proportion of peripheral Th17 cells and LFA-1 surface expression in MPTP-treated mice, and Th17 cell levels in the SN also significantly decreased. CCL5, which increased Th17 cell differentiation and LFA-1 protein expression by activating LCK and ZAP70, could increase the Th17 cell number in the SN, induce DA neuron death and aggravate PD.

Not all (cells) who wander are lost: Upstream migration as a pervasive mode of amoeboid cell motility.

Leukocytes possess the ability to migrate upstream-against the direction of flow-on surfaces of specific chemistry. Upstream migration was first characterized in vitro for T-cells on surfaces comprised of intracellular adhesion molecule-1 (ICAM-1). Upstream migration occurs when the integrin receptor αLβ2 (also known as lymphocyte function-associated antigen-1, or LFA-1) binds to ICAM-1. LFA-1/ICAM-1 interactions are ubiquitous and are widely found in leukocyte trafficking. Upstream migration would be employed after cells come to arrest on the apical surface of the endothelium and might confer an advantage for both trans-endothelial migration and tissue surveillance. It has now been shown that several other motile amoeboid cells which have the responsibility of trafficking from blood vessels into tissues, such as Marginal zone B cells, hematopoietic stem cells, and neutrophils (when macrophage-1 antigen, Mac-1, is blocked), can also migrate upstream on ICAM-1 surfaces. This review will summarize what is known about the basic mechanisms of upstream migration, which cells have displayed this phenomenon, and the possible role of upstream migration in physiology and tissue homeostasis.

Virtual Screening and Binding Analysis of Potential CD58 Inhibitors in Colorectal Cancer (CRC).

Human cell surface receptor CD58, also known as lymphocyte function-associated antigen 3 (LFA-3), plays a critical role in the early stages of immune response through interacting with CD2. Recent research identified CD58 as a surface marker of colorectal cancer (CRC), which can upregulate the Wnt pathway and promote self-renewal of colorectal tumor-initiating cells (CT-ICs) by degradation of Dickkopf 3. In addition, it was also shown that knockdown of CD58 significantly impaired tumor growth. In this study, we developed a structure-based virtual screening pipeline using Autodock Vina and binding analysis and identified a group of small molecular compounds having the potential to bind with CD58. Five of them significantly inhibited the growth of the SW620 cell line in the following in vitro studies. Their proposed binding models were further verified by molecular dynamics (MD) simulations, and some pharmaceutically relevant chemical and physical properties were predicted. The hits described in this work may be considered interesting leads or structures for the development of new and more efficient CD58 inhibitors.

LFA-1 knockout inhibited the tumor growth and is correlated with treg cells

Cancer immunotherapy has been proven to be clinically effective in multiple types of cancers. Lymphocyte function-associated antigen 1 (LFA-1), a member of the integrin family of adhesion molecules, is expressed mainly on αβ T cells. LFA-1 is associated with tumor immune responses, but its exact mechanism remains unknown. Here, two kinds of mice tumor model of LFA-1 knockout (LFA-1−/−) mice bearing subcutaneous tumor and ApcMin/+;LFA-1−/− mice were used to confirm that LFA-1 knockout resulted in inhibition of tumor growth. Furthermore, it also demonstrated that the numbers of regulatory T cells (Treg cells) in the spleen, blood, mesenteric lymph nodes were decreased in LFA-1−/− mice, and the numbers of Treg cells in mesenteric lymph nodes were also decreased in ApcMin/+;LFA-1−/− mice compared with ApcMin/+ mice. LFA-1 inhibitor (BIRT377) was administered to subcutaneous tumor-bearing LFA-1+/+ mice, and the results showed that the tumor growth was inhibited and the number of Treg cells was reduced. The analysis of TIMER tumor database indicated that LFA-1 expression is positively associated with Treg cells and TNM stage. Conclusively, this suggests that LFA-1 knockout would inhibit tumor growth and is correlated with Treg cells. LFA-1 may be one potential target for cancer immunotherapy.62QPUkCarP1aXjSsQjtmWhVideo

Establishment of an LC-MS/MS method for quantification of lifitegrast in rabbit plasma and ocular tissues and its application to pharmacokinetic study

Lifitegrast, a lymphocyte function-associated antigen 1 antagonist, was approved by the FDA for the treatment of dry eye disease. Cornea and conjunctiva have been reported to be the sites of action of lifitegrast. To investigate the pharmacokinetics of lifitegrast, a sensitive analytical method for the determination of lifitegrast in various biological matrices such as plasma and ocular tissues is required. However, only limited information about the analytical method for lifitegrast in biological samples is available. In the present study, we aimed to develop a new liquid chromatography-tandem mass spectrometry method for the determination of lifitegrast in rabbit plasma, cornea, conjunctiva, and sclera. Lifitegrast-d6 was used as an internal standard (IS). To prepare the biological samples, protein precipitation using acetonitrile was utilized. Analytes were separated from endogenous interferences on an Atlantis dC18 (5 µm, 2.1 × 150 mm), and a mixture of 0.1 % formic acid and acetonitrile was used as the mobile phase. The mass transition of precursor to product ion was monitored at 615.2 → 145.0 for lifitegrast and 621.2 → 145.1 for IS. The calibration curves were linear over the concentration range from 2 to 500 ng/mL for plasma and 5 to 500 ng/mL in ocular tissue homogenates. Intra- and inter-day accuracy ranged from 95.76 to 106.80 % in the plasma and 94.42 to 112.80 % in the ocular tissues. Precision was within 8.56 % in the plasma and 9.72 % in the ocular tissues. The short-term, long-term, auto-sampler, and freeze–thaw stabilities of lifitegrast were validated. The developed method was applied to a pharmacokinetic study of lifitegrast in rabbits. Following ophthalmic administration, only 3.26 % of administered lifitegrast was absorbed into the systemic circulation. Peak tissue concentrations were observed at 0.5 h after dosing, and topically administered lifitegrast was mainly distributed in the cornea and conjunctiva. The finding of this study is expected to be used in further pharmacokinetic studies and formulation development.