Expression Of LFA-1 Research Articles

Effects of HTLV-1 on leukocyte trafficking and migration in ACs compared to healthy individuals

Human T-lymphotropic virus type 1 (HTLV-1) is a RNA virus belonging to Retroviridae family and is associated with the development of various diseases, including adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Aside from HAM/TSP, HTLV-1 has been implicated in the development of several disorders that mimic auto-inflammation. T-cell migration is important topic in the context of HTLV-1 associated diseases progression. The primary objective of this case–control study was to assess the relationship between increased mRNA expression in virus migration following HTLV-1 infection. PBMCs from 20 asymptomatic patients and 20 healthy subjects were analyzed using real-time PCR to measure mRNA expression of LFA1, MLCK, RAC1, RAPL, ROCK1, VAV1 and CXCR4. Also, mRNA expression of Tax and HBZ were evaluated. Mean expression of Tax and HBZ in ACs (asymptomatic carriers) was 0.7218 and 0.6517 respectively. The results revealed a noteworthy upregulation of these genes involved in T-cell migration among ACs patients in comparison to healthy individuals. Considering the pivotal role of gene expression alterations associated with the progression into two major diseases (ATLL or HAM/TSP), analyzing the expression of these genes in the ACs group can offer probable potential diagnostic markers and aid in monitoring the condition of ACs.

IL-10 indirectly modulates functional activity of CD4+CD28null T-lymphocytes through LFA-3 and HLA class II inhibition.

Expansion of CD4+CD28null T-lymphocytes is common in chronic heart failure (CHF) patients. Its ability to produce high levels of proinflammatory cytokines is probably the key role of these cells in CHF. IL-10 is a candidate for limiting CD4+CD28null T-lymphocyte responses, whereas tumour necrosis factor (TNF) is the cytokine most closely involved in the loss of CD28 expression. Serum levels of TNF and IL-10 were measured in 65 CHF patients (mean age, 65.2 ± 13.84 years). Patients with an IL-10/TNF ratio ≥1 had significantly lower levels of CD4+CD28null T-lymphocytes than those with a ratio <1. In vitro, IL-10 reduced the frequency of proliferative CD4+CD28null T-lymphocytes stimulated with anti-CD3. Pre-treatment with IL-10 before anti-CD3 stimulation was required for the cytokine to inhibit TNF production by CD4+CD28null T-lymphocytes. In addition to the previously described effect of IL-10 on HLA-DR and ICAM-1 expression, LFA-3 protein and mRNA levels were reduced in the presence of the cytokine in monocytes. IL-10 inhibition on CD4+CD28null T-lymphocytes may be mediated by a reduction in HLA class II and LFA-3 expression because blocking interactions with these costimulators has similar effects to those of IL-10 treatment. Moreover, costimulation through CD2/LFA-3 interaction is enough to induce proliferation and cytokine production in CD4+CD28null T-lymphocytes.

Ets1 represses a T-bet dependent migration program and stabilizes the iNKT1 adhesome

Abstract iNKT cells are innate-like T cells that recognize glycolipids presented on MHCI-like CD1d using a semi-invariant TCR. Upon activation, iNKTs rapidly produce cytokines to direct the adaptive response towards a T H1, T H2 or T H17 response. The transcription factor ETS1 is important for iNKT cell development, but its roles in iNKT function and homeostasis are not well characterized. Utilizing a novel mouse model for targeted deletion of Ets1 in iNKT1s, we show that ETS1 limits expression of T-bet, the signature transcription factor of the iNKT1 lineage. Ets1 Δ/Δhepatic iNKT1 cells upregulated a gene program associated with migration and had reduced expression of LFA-1 and ICAM-1, receptors critical for retention in the liver. Remarkably, Ets1 Δ/Δliver iNKT1s remained tissue resident but no longer required LFA-1/ICAM-1 for retention. Heterozygous deletion of T-bet abrogated expression of multiple migration-associated genes, restored LFA-1 and ICAM-1, and re-established reliance on LFA-1/ICAM-1 for retention. Our data demonstrate that ETS1 controls the mechanisms of hepatic iNKT1 cell residency by limiting a T-bet-dependent migration and adhesion program. Supported by NIH/NIAID grant R01 AI106352

Investigation of the clinical utility of adhesion molecules in the management of thyroid nodules

To better understand the relationship among cell adhesion molecules (CAM) and investigate the clinical diagnostic and prognostic application of ICAM-1 (ICAM1), LFA-1 (ITGAL), and L-selectin (SELL) proteins and mRNA corresponding expression in thyroid cancer. Gene expression was evaluated by RT–qPCR, and protein expression was evaluated by immunohistochemistry. We evaluated 275 patients (218 women, 57 men, 48.4 ± 14.5 years old), including 102 benign and 173 malignant nodules. The 143 papillary thyroid carcinoma (PTC) and 30 follicular thyroid carcinoma (FTC) patients were managed according to current guidelines and followed-up for 78.7 ± 54.2 months. Malignant and benign nodules differed concerning mRNA (p = 0.0027) and protein (p = 0.0020 for nuclear) expression of L-selectin and ICAM-1 (mRNA: p = 0.0001 and protein: p = 0.0014) and protein expression of LFA-1 (p = 0.0168), but not mRNA expression of LFA-1 (p = 0.2131). SELL expression was more intense in malignant tumors (p = 0.0027). ICAM1 (p = 0.0064) and ITGAL (p = 0.0244) mRNA expression was higher in tumors with lymphocyte infiltrate. ICAM-1 expression correlated with younger age at diagnosis (p = 0.0312) and smaller tumor size (p = 0.0443). Also, LFA-1 expression correlated with higher age at diagnosis (p = 0.0376) and was more intense at stage III and IV (p = 0.0077). In general, the protein expression of the 3 CAM decreased as the process of cellular dedifferentiation occurred. We suggest that the SELL and ICAM1 genes and L-selectin and LFA-1 protein expression may help confirm malignancy and assist in the histological characterization of follicular patterned lesions, but we were unable to correlate these CAMs with patient outcomes.

Exploration of immunosuppressive features of the tumor microenvironment within hepatic and non-hepatic tumors of urothelial origin.

562 Background: Recent data suggest that patients with liver metastases (mets) are resistant to immune checkpoint inhibition (CPI) independent of historical biomarkers of CPI efficacy, raising the question of whether liver mets may be associated with an immunosuppressive tumor microenvironment (TME). We investigated the immune TME of hepatic and non-hepatic mets compared to primary tumors in advanced urothelial carcinoma (UC) tissue samples. Methods: NextGen sequencing (NGS) of DNA (592-gene/whole exome) and RNA (whole transcriptome) from UC tissue samples (N=4746) was performed at Caris Life Sciences (Phoenix, AZ). Immune cell infiltration was estimated by RNA expression deconvolution (MCP-counter). PD-L1 expression (SP142: immune cell stain ≥ 5%; 22c3: CPS ≥ 10) was assessed by immunohistochemistry (IHC). Deficient mismatch repair/high microsatellite instability (dMMR/MSI-H) was tested by IHC/NGS. Real-world overall survival (OS) information was obtained from insurance claims data and Kaplan-Meier estimates were calculated. Mann–Whitney U and X2/Fischer-Exact tests were applied where appropriate, with p-values adjusted for multiple comparisons (Benjamini-Hochberg). *P&lt;0.05. Results: UC samples included 3158 (66.5%) from primary site, 1344 (28.3%) from non-hepatic mets, and 244 (5.1%) from hepatic mets. Compared to primary tumors, hepatic mets had decreased CD8+ T and B cells (0.55* and 0.29-fold*) but increased monocytic lineage cells (1.23-fold*), while non-hepatic mets had increased CD8+ T, NK, and monocytic lineage cells (1.28*, 1.27*, 1.31-fold*) with no difference in B cells (1.05-fold). Hepatic mets had decreased expression of integrin LFA-1/ ITGAL (0.77-fold*), as well as hyaluronic acid (HA) receptor CD44 and synthase HAS2 (0.61 and 0.61-fold*), compared to primary tumors, whereas expression of these genes and LFA-1 ligand ICAM1 was increased in non-hepatic mets (1.08 to 1.30-fold*). Hepatic mets had increased expression of immunosuppressive cytokines CCL2 and CXCL2 (1.72* and 2.32-fold*) and decreased expression of pro-inflammatory cytokines CCL5 and CXCL10 (0.63* and 0.79-fold*) compared to primary tumors. PD-L1+ IHC was less frequent in hepatic mets compared to primary tumors and non-hepatic mets. TMB-High (≥10 mut/MB) and dMMR/MSI-H rates were similar across tumor sites. Hepatic mets (N=40) were associated with worse OS from the start of pembrolizumab compared to non-hepatic mets (N=177) (19.6 vs 4.4 months, HR 3.01, 95% CI 1.91-4.75, p&lt;0.0001). Conclusions: This is the largest analysis of hepatic and non-hepatic met TMEs compared to primary tumor in advanced UC. Lower PD-L1 expression and differences in immune TME composition in liver mets may contribute to CPI resistance. Further analysis is warranted to determine underlying molecular mechanisms resulting in a TME that reduces response to CPI for patients with UC and liver mets.

Impaired Treg-DC interactions contribute to autoimmunity in leukocyte adhesion deficiency type 1.

Leukocyte adhesion deficiency type 1 (LAD-1) is a rare disease resulting from mutations in the gene encoding for the common β-chain of the β2-integrin family (CD18). The most prominent clinical symptoms are profound leukocytosis and high susceptibility to infections. Patients with LAD-1 are prone to develop autoimmune diseases, but the molecular and cellular mechanisms that result in coexisting immunodeficiency and autoimmunity are still unresolved. CD4+FOXP3+ Treg are known for their essential role in preventing autoimmunity. To understand the role of Treg in LAD-1 development and manifestation of autoimmunity, we generated mice specifically lacking CD18 on Treg (CD18Foxp3), resulting in defective LFA-1 expression. Here, we demonstrate a crucial role of LFA-1 on Treg to maintain immune homeostasis by modifying T cell-DC interactions and CD4+ T cell activation. Treg-specific CD18 deletion did not impair Treg migration into extralymphatic organs, but it resulted in shorter interactions of Treg with DC. In vivo, CD18Foxp3 mice developed spontaneous hyperplasia in lymphatic organs and diffuse inflammation of the skin and in multiple internal organs. Thus, LFA-1 on Treg is required for the maintenance of immune homeostasis.

Formyl peptide receptors are involved in CTX-induced impairment of lymphocyte functions

Crotoxin (CTX) is a neurotoxin that is isolated from the venom of Crotalus durissus terrificus, which displays immunomodulatory, anti-inflammatory, and anti-tumoral effects. Previous research has demonstrated that CTX promotes the adherence of leukocytes to the endothelial cells in blood microcirculation and the high endothelial venules of lymph nodes, which reduces the number of blood cells and lymphocytes. Studies have also shown that these effects are mediated by lipoxygenase-derived mediators. However, the exact lipoxygenase-derived eicosanoid involved in the CTX effect on lymphocytes is yet to be characterized. As CTX stimulates lipoxin-derived mediators from macrophages and lymphocyte effector functions could be modulated by activating formyl peptide receptors, we aimed to investigate whether these receptors were involved in CTX-induced redistribution and functions of lymphocytes in rats. We used male Wistar rats treated with CTX to demonstrate that Boc2 (butoxycarbonyl-Phe-Leu-Phe-Leu-Phe), an antagonist of formyl peptide receptors, prevented CTX-induced decrease in the number of circulating lymphocytes and increased the expression of the lymphocyte adhesion molecule LFA1. CTX reduced the T and B lymphocyte functions, such as lymphocyte proliferation in response to the mitogen Concanavalin A and antibody production in response to BSA immunization, respectively, which was prevented by the administration of Boc2. Importantly, mesenteric lymph node lymphocytes from CTX-treated rats showed an increased release of 15-epi-LXA4. These results indicate that formyl peptide receptors mediate CTX-induced redistribution of lymphocytes and that 15-epi-LXA4 is a key mediator of the immunosuppressive effects of CTX.

Unveiling Highly Fluctuating Single-Cell Transcriptome Dynamics at a Novel Bifurcation Point in a Human Lymphoid Pathway

Background: Human hematopoiesis was considered a hierarchical stepwise process, but on the basis of recent progress in single-cell RNA-sequencing (scRNA-seq) it is now considered a continuous process. However, the uncertainty or fluctuation of single-cell transcriptome dynamics during differentiation was not considered. To address this issue, we developed a new computational methodology that converts single-cell splicing kinetics into an advection-diffusion model in a latent cell state using deep learning technology, which enabled us to quantitatively capture the fluctuation in cell state dynamics. Because the human lymphoid pathway and bifurcation points for dendritic cells (DC)/monocytic lineage remain incompletely defined, we applied our method to explore the lymph-DC pathway in the human lympho-hematopoietic system. Materials and methods: CD34+ or CD34+CD38+CD45RA+CD19-CD7-CD10+ cells (CD10SPs) were sorted from a mixture of frozen human cord blood samples, and scRNA-seq and single-cell chromatin accessibility analysis (scATAC) were performed. The data were applied to the variational inference of cell state dynamics with fluctuation (VICDYF). Subsequently, the obtained findings were validated by novel mesenchymal stem cell (MSC)-like coculture system that supports comprehensive human hematopoiesis. Results: MSC-like cultures of various lymphoid populations showed that the CD10SP population, which had been thought to be B/NK progenitors, was a heterogenous population in which lineage diversification from multi-lymphoid progenitors toward lymphoids and DCs was suspected to occur. Our insilico analysis of CD34+ cells with VICDYF suggested a B/plasmacytoid DC (pDC) bifurcation with high fluctuating transcriptome dynamics in the CD10-expressing fraction among various levels of fluctuating bifurcation points. Analysis of CD10SPs identified the B/pDC bifurcation as a highly fluctuating transcriptome state in the IL-7Rα+ fraction in association with the LFA-1 expression dynamics and pDC differentiation bias. Consistent with these findings, B/pDC common progenitors were identified by single cell culture, and the LFA-1-high population gave rise to a higher percentage of pDCs than proB cells, while the LFA-1-low population produced a higher percentage of proB cells than pDCs in the c-kit+IL-7Rα+ fraction. Of note, the epigenetic regulator SFMBT2 was specifically expressed in the B/pDC bifurcation region. The scATAC of the CD34+ population revealed that the B/pDC bifurcation with large fluctuation coincided with the high accessibility of both sides of the differentially accessible chromatin regions for both B and pDC lineages. This study revealed a new landscape of the human lymphoid pathway that includes crucial bifurcation points with highly fluctuating single-cell transcriptome dynamics. Discussion: Our computational methodology discovered a novel B/pDC bifurcation as high fluctuating dynamics. The insilico predictions were confirmed by the biological findings, using MSC-like cultures. Although the underlying molecular mechanism remains elusive, single-cell multiomics analysis suggests that the estimated large fluctuation is associated with epigenetic changes in chromatin regions for both B and pDC lineages. VICDYF also identified the highly fluctuated transcriptome dynamics at the putative lineage bifurcation point during pancreatic development. Conclusion: Our results provide a new model of differentiation - fluctuation based differentiation - which reconciles continuous and stepwise models. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CBA strain mice as a model of spontaneous carcinogenesis

The review analyzes some parameters of CBA mice-males as model of spontaneous carcinogenesis characterizing adhesive and adaptive disorders. A weakening of the hepatocytes mutual adhesiveness force was noted already in early ontogenesis (5–10 days of postnatal development). This violation persisted and enhanced during hepatocarcinogenesis. A decrease of the β2 leukocyte integrins LFA-1 and Mac-1 expression on peripheral blood cells as well as an increase of the interleukins 6 and 10 in blood serum were determined during ontogenesis. It is significant for weakening the liver cells contact interactions (mutual adhesiveness) as well as immunity effectors and tumor cells interactions. A disbalance of the adaptive reactions and life quality important components was revealed in the CBA mice-males ontogenesis. Number of dopaminergic neurons and the neurogenesislevel in CBA micemales were decreasing. This does not contradict the dynamics of chronic stress and the aging process: an increase in the catabolic stress hormone corticosterone, a decrease in the anabolic hormone testosterone in the blood serum, a decrease in motor activity, signs of cachexia and alopecia, as well as a violation of immunological parameters.CBA mice-males with an assessment of parameters characterizing adhesive and adaptive disorders during spontaneous carcinogenesis (the hepatocytes mutual adhesiveness forсe, the expression of β2 leukocyte integrins LFA-1 and Mac-1 on peripheral blood cells, the content of interleukins 6, 10, corticosterone and testosterone in blood serum, the number of dopaminergic neurons in the midbrain during ontogenesis) as well as the frequency and size of tumours, lifespan and somatic status of animals can be used as a scientifically- and evidence-based test system to study cytostatic drugs as well as non-toxic geroprotective medications for prevention and treatment of cancer in individuals with an increased risk of malignant neoplasms developing especially hepatocellular carcinoma.

LFA-1/ ICAM-1 promotes NK cell cytotoxicity associated with the pathogenesis of ocular toxoplasmosis in murine model.

Ocular toxoplasmosis (OT) is one of the most common causes of posterior uveitis. However, the pathogenic mechanisms of OT have not been well elucidated. Here, we used C57BL/6 (B6) mice to establish OT by peroral infection with 20 cysts of the TgCtWh6 strain, and severe ocular damage was observed by histopathological analysis in the eyes of infected mice. RNA-sequencing results showed that infection with T. gondii increased the expression of the NK-mediated cytotoxicity gene pathway at Day 30 after ocular T. gondii infection. Both NK-cell and CD49a+ NK-cell subsets are increased in ocular tissues, and the expression levels of LFA-1 in NK cells and ICAM-1 in the OT murine model were upregulated upon infection. Furthermore, inhibition of the interaction between LFA-1 and ICAM-1 with lifitegrast, a novel small molecule integrin antagonist, inhibited the protein expression of LFA-1 and ICAM-1 in murine OT and NK cells, improved the pathology of murine OT and influenced the secretion of cytokines in the OT murine model. In conclusion, the interaction between LFA-1 and ICAM-1 plays a role in the early regulation of the CD49a+ NK-cell proportion in an OT murine model. LFA-1/ ICAM-1 may be a key molecule in the pathogenesis of OT, and may provide new insights for potential immunotherapy.

Antiphospholipid Antibody Syndrome-Associated Increased Surface Expression of VLA4 Integrin on Human Monocytes.

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by thrombosis and/or obstetric complications in the presence of antiphospholipid antibodies (aPL). Catastrophic APS (CAPS) is the most severe form of the disease, in which microvascular thromboses develop rapidly, leading to multiorgan failure. Monocytes, along with endothelial cells, are critical players in the pathogenesis of APS. Recruitment of these cells to the site of injury/inflammation involves a series of events, including capture, rolling, adhesion enhancement, and transmigration, which are controlled by surface adhesion molecules. The aim of our study was to investigate the surface adhesion profile of monocytes from APS patients and monocytes stimulated in vitro with aPL from a CAPS patient. The surface expression of the adhesion molecules LFA1, L-selectin, MAC1, PSGL1, and VLA4 was analyzed by flow cytometry. To our knowledge, this preliminary study was the first to show that VLA4 was significantly increased on the surface of monocytes from APS patients. Moreover, in vitro stimulations mimicking CAPS showed an even greater increase in VLA4. Our data suggest that the surface adhesion profile on monocytes is altered in APS and CAPS and may be involved in the thrombotic pathophysiology of the disease by enhancing monocyte adhesion.

A bifurcation concept for B-lymphoid/plasmacytoid dendritic cells with largely fluctuating transcriptome dynamics.

Hematopoiesis was considered a hierarchical stepwise process but was revised to a continuous process following single-cell RNA sequencing. However, the uncertainty or fluctuation of single-cell transcriptome dynamics during differentiation was not considered, and the dendritic cell (DC) pathway in the lymphoid context remains unclear. Here, we identify human B-plasmacytoid DC (pDC) bifurcation as large fluctuating transcriptome dynamics in the putative B/NK progenitor region by dry and wet methods. By converting splicing kinetics into diffusion dynamics in a deep generative model, our original computational methodology reveals strong fluctuation at B/pDC bifurcation in IL-7Rα+ regions, and LFA-1 fluctuates positively in the pDC direction at the bifurcation. These expectancies are validated by the presence of B/pDC progenitors in the IL-7Rα+ fraction and preferential expression of LFA-1 in pDC-biased progenitors with a niche-like culture system. We provide a model of fluctuation-based differentiation, which reconciles continuous and discrete models and is applicable to other developmental systems.

Galectin-9 supports primary T cell transendothelial migration in a glycan and integrin dependent manner

Adaptive immunity relies on the efficient recruitment of T cells from the blood into peripheral tissues. However, the current understanding of factor(s) coordinating these events is incomplete. Previous studies on galectin-9 (Gal-9), have proposed a functionally significant role for this lectin in mediating leukocyte adhesion and transmigration. However, very little is known about its function in T cell migration. Here, we have investigated the role of the Gal-9 on the migration behaviour of both human primary CD4+ and CD8+ T cells. Our data indicate that Gal-9 supports both CD4+ and CD8+ T cell adhesion and transmigration in a glycan dependent manner, inducing L-selectin shedding and upregulation of LFA-1 and CXCR4 expression. Additionally, when immobilized, Gal-9 promoted capture and firm adhesion of T cells under flow, in a glycan and integrin-dependent manner. Using an in vivo model, dorsal air pouch, we found that Gal-9 deficient mice display impaired leukocyte trafficking, with a reduction in pro-inflammatory cytokines/chemokines generated locally. Furthermore, we also demonstrate that Gal-9 inhibits the chemotactic function of CXCL12 through direct binding. In conclusion, our study characterises, for the first time, the capture, adhesion, and migration behaviour of CD4+ and CD8+ T cells to immobilised /endothelial presented Gal-9, under static and physiological flow conditions. We also demonstrate the differential binding characteristics of Gal-9 to T cell subtypes, which could be of potential therapeutic significance, particularly in the treatment of inflammatory-based diseases, given Gal-9 ability to promote apoptosis in pathogenic T cell subsets.

A new β2 integrin activation reporter mouse reveals localized intra‐ and extra‐vascular neutrophil integrin activation in vivo

β2 integrins (LFA‐1, Mac‐1, CD11c‐CD18, and CD11d‐CD18) are leukocyte‐specific adhesion receptors that play critical roles in leukocyte recruitment, as well as other immunological processes such as phagocytosis and immunological synapse formation between T cells and antigen presenting cells. Adhesion of leukocytes to other cells such as endothelial cells are regulated by integrin affinity changes for their ligands (“activation”). For human β2 integrins, activation reporter antibodies including mAb24 and KIM127 can be used to study integrin activation. No such activation epitopes are known in mouse β2 integrins. Because of the lack of mouse β2 integrin activation reporter antibodies, nothing is known about β2 integrin activation in vivo. Here, we generated a humanized β2 integrin knockin mouse strain by targeting the human β2 integrin coding sequence into the mouse Itgb2 locus. We show that this enables imaging of β2 integrin activation using the KIM127 (extension conformation) and mAb24 (high affinity) reporter antibodies. Human β2 pairs with the mouse integrin α chains, yielding normal expression of the β2 integrins LFA‐1, Mac‐1 and CD11c‐CD18 in all major leukocyte populations. Using a CXCL1‐induced acute inflammation model, we uncovered the dynamics and subcellular localization of β2 integrin activation in arresting neutrophils in vivo in venules of the mouse cremaster muscle. Activated integrins in arresting neutrophils in vivo are concentrated at the interface of neutrophils and the endothelium at the rear side of neutrophils facing against the blood flow. In a high‐dose lipopolysaccharide (LPS) model, we found that β2 integrins are activated in association with elevated neutrophil adhesion in lung and liver. Thus, these mice, for the first time, enable studies into β2 integrin activation in vivo.

Mechanosensory channel Piezo1 contributes to T lymphocyte migration through direct interaction with integrins

Abstract Response to mechanical signals is an important mechanism by which T lymphocytes drive inflammation. A potential contributor in this process is Piezo1, a mechanosensitive cation channel which is abundantly expressed in T lymphocytes. Previously, our lab has shown that Piezo1 colocalizes with high affinity LFA-1 on activated human T lymphocytes during chemokine-activated crawling, suggesting a role for Piezo1 in the adhesion cascade. However, the mechanism by which Piezo1 contributes to the regulation of the adhesion cascade is unknown. We hypothesize that Piezo1 mediates T lymphocyte migration through direct contact with integrin to regulate its function. We investigate the role of Piezo1 in the expression, binding affinity, and turnover of LFA-1 and VLA-4. Co-immunoprecipitation revealed a preferential association of Piezo1 with the alpha subunit of integrins, specifically LFA-1, MAC-1, and VLA-4 in the active forms. Moreover, microfluidic devices were used to study the contribution of Piezo1 on T lymphocytes under laminar flow-induced shear stress. We found that activated T lymphocytes with CRISPR/Cas9-mediated Piezo1 ablation resulted in a reduced chemokine-induced crawling speed that correlates to the efficiency of Piezo1 gene ablation. Finally, in studies of lymph node homing where fluorescently labeled T lymphocytes were tracked in vivo, Piezo1 deficient cells exhibit increased ingress and decreased egress through the lymph node, suggesting an intricate role of Piezo1 on T lymphocyte migration. Taken together, our data support the hypothesis that Piezo1 modulates inflammatory processes through complex integrin interactions and mediates T lymphocyte migration from the lymph node to the site of inflammation. Case Postbaccalaureate Research Education Program grant NIH/NIGMS 5R25GM075207-15; St. Baldrick’s Foundation; Hyundai Hope-on-Wheels Program; The I’m Not Done Yet Foundation; and Pediatric Cancer Research Foundation

A new β2 integrin activation reporter mouse reveals localized intra- and extra-vascular neutrophil integrin activation in vivo

Abstract β2 integrins (LFA-1, Mac-1, CD11c-CD18, and CD11d-CD18) are leukocyte-specific adhesion receptors that play critical roles in leukocyte recruitment, as well as other immunological processes such as phagocytosis and immunological synapse formation. Adhesion of leukocytes to other cells such as endothelial cells are regulated by integrin affinity changes for their ligands (“activation”). Human β2 integrin activation can be detected by reporter antibodies including mAb24 and KIM127. No such activation epitopes are known in mouse β2 integrins. Because of the lack of mouse β2 integrin activation reporter antibodies, nothing is known about β2 integrin activation in vivo. Here, we generated a humanized β2 integrin knockin mouse by targeting the human β2 integrin coding sequence into the mouse Itgb2 locus. We show that this enables imaging of β2 integrin activation using the KIM127 (extension conformation) and mAb24 (high affinity) reporter antibodies. Human β2 pairs with the mouse integrin α chains, yielding normal expression of the β2 integrins LFA-1, Mac-1 and CD11c-CD18 in all major leukocyte populations. Using a CXCL1-induced acute inflammation model, we uncovered the dynamics and subcellular localization of β2 integrin activation in arresting neutrophils in vivo in venules of the mouse cremaster muscle. Activated integrins in arresting neutrophils in vivo are concentrated at the interface of neutrophils and the endothelium at the rear side of neutrophils facing against the blood flow. In a high-dose lipopolysaccharide (LPS) model, we found that β2 integrins are activated in association with elevated neutrophil adhesion in lung and liver. Thus, these mice, for the first time, enable studies into β2 integrin activation in vivo. This work was supported by grants from the National Institutes of Health, USA (HL078784 to K.L.) and a Postdoctoral Fellowship (19POST34450228 to L.W.) from the American Heart Association, USA.

Dendritic cell activation by a micro particulate based system containing the influenza matrix-2 protein virus-like particle (M2e VLP)

M2e VLP was previously described as a vaccine that incorporates the extracellular region of the matrix 2 protein (M2e), which is highly conserved amongst all the strains of influenza. In this study, we analyzed activation status of dendritic cells (DCs) after exposure to M2e VLP, stimulating DCs with M2e VLP and co-culturing the stimulated DCs with T cells to observe innate and adaptive immune responses. The M2e VLP microparticle was prepared by encapsulating into a polymer matrix using the one-step spray drying method. Adjuvants Alhydrogel®, MPL-A® or AddavaxTM were used to enhance the DC stimulatory effects by the M2e VLP microparticle. The M2e VLP microparticle yield was found to be 92% and the encapsulation yield was around 84% with a size of approximately 2.78 μm. There was no short-term cytotoxicity found in DCs and macrophages with concentrations up to 1500 μg/mL of M2e VLP microparticle, however long-term exposure resulted in 25% decrease in viability of cells with concentrations more than or equal to 500 μg/mL. The M2e VLP microparticle vaccine with Alhydrogel® and MPL-A® induced high levels of TNFα in both DCs and macrophages. The high levels of MHC I, II, CD28, B7-1, ICAM-1, LFA-1 expression and IL-12 release in the M2e VLP microparticle group with Alhydrogel® suggests that the M2e VLP vaccine with this adjuvant activated T cells via the Th2 pathway. The increased expression of MHC I, II, CD40, CD154, ICAM-1 and LFA-1 on DCs and the release of IL-12 in the M2e VLP microparticle culture of DCs with MPL-A® demonstrated that the M2e VLP vaccine with this adjuvant activated T cells via the Th1 pathway. The decrease in fluorescence in the Alhydrogel® and MPL-A® group illustrates the proliferation of T cells took place following exposure of DCs to the M2e VLP microparticle with these adjuvants. The M2e VLP microparticle exhibited higher stimulatory responses of DCs than the M2e VLP in suspension. Furthermore, the presence of Alhydrogel® and MPL-A® enhanced the stimulatory effects of DCs by the M2e VLP microparticle (MP) vaccine.

Erxian Decoction modulates Th17/Treg cells differentiation through LFA-1/ICAM-1/STAT3 pathway in menopausal dry eye disease

With the development of modern societies and the ageing of the population, the treatment of menopausal dry eye disease (MDED) has become a thorny issue for the medical profession. Erxian Decoction (EXD) is a traditional Chinese medicine prescription, which has performed good clinical effect on dry eye disease. In this research, we purposed to investigate the molecular mechanisms of EXD for the treatment of MDED. A MDED rat model was established, the results indicated that high concentration of EXD could significantly improve the tear secretion and tear film stability of the animal model. Next, we found that EXD worked through the LFA-1/ICAM-1/STAT3 pathway in the body, and EXD could regulate IL-17, IL-10, CTLA-4 and TGF-β1 to get Th17/Treg balance. In vitro experiments, the results indicated that EXD affected the differentiation of CD4+ T cells into Th17/Treg cells by inhibiting the expression and activation of LFA-1 on CD4+ T cells, thus exerting immunotherapy effect. Our research provided the experimental basis and associated mechanisms for the clinical application of EXD in dry eye disease.

Deficiency of Leukocyte Surface Glycoproteins Mol, LFA-1, and Leu M5 in a Dog With Recurrent Bacterial Infections: An Animal Model

A dog with severe recurrent bacterial infections, impaired pus formation, delayed wound healing, and severe persistent leukocytosis was the result of a mother-son mating. Assessment of leukocyte function revealed profound abnormalities in adherence-dependent activities including impaired granulocyte adhesion to glass/plastic surfaces or nylon wool, decreased granulocyte aggregation and chemotaxis, and diminished lymphocyte blastogenesis, but normal neutrophil oxidative activity, serum immunoglobulin, and complement levels. By immunofluorescence analysis, CD11b and CD18 monoclonal antibodies specific for the 155-kd alpha polypeptide of Mo1 (gp 155, 94) and the 94 kd beta peptide common to Mo1, LFA-1 (gp 170, 94), and Leu M5 (p 150, 94) (surface molecules that promote leukocyte adhesion) failed to bind to unstimulated and A23187 calcium ionophore-stimulated granulocytes or mononuclear cells of the affected dog as compared with strong specific binding to canine control cells. The Mo1 glycoproteins were only barely detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of immunoprecipitates from lysates of 125I surface-labeled neutrophils from the affected dog as compared with intense bands seen with canine control cell precipitates. We conclude that this dog has a severe leukocyte surface glycoprotein deficiency syndrome that is similar, if not identical, to that recently recognized in humans. Dogs with deficiency of leukocyte Mo1, LFA-1, and Leu M5 expression may represent a useful animal model to characterize further the molecular basis for an inherited disorder in leukocyte effector function.

Cladribine Alters Immune Cell Surface Molecules for Adhesion and Costimulation: Further Insights to the Mode of Action in Multiple Sclerosis.

Cladribine (CLAD) is a deoxyadenosine analogue prodrug which is given in multiple sclerosis (MS) as two short oral treatment courses 12 months apart. Reconstitution of adaptive immune function following selective immune cell depletion is the presumed mode of action. In this exploratory study, we investigated the impact of CLAD tablets on immune cell surface molecules for adhesion (CAMs) and costimulation (CoSs) in people with MS (pwMS). We studied 18 pwMS who started treatment with CLAD and 10 healthy controls (HCs). Peripheral blood mononuclear cells were collected at baseline and every 3 months throughout a 24-month period. We analysed ICAM-1, LFA-1, CD28, HLADR, CD154, CD44, VLA-4 (CD49d/CD29), PSGL-1 and PD-1 with regard to their expression on B and T cells (T helper (Th) and cytotoxic T cells (cT)) and surface density (mean fluorescence intensity, MFI) by flow cytometry. The targeted analysis of CAM and CoS on the surface of immune cells in pwMS revealed a higher percentage of ICAM-1 (B cells, Th, cT), LFA-1 (B cells, cT), HLADR (B cells, cT), CD28 (cT) and CD154 (Th). In pwMS, we found lower frequencies of Th and cT cells expressing PSGL-1 and B cells for the inhibitory signal PD-1, whereas the surface expression of LFA-1 on cT and of HLADR on B cells was denser. Twenty-four months after the first CLAD cycle, the frequencies of B cells expressing CD44, CD29 and CD49d were lower compared with the baseline, together with decreased densities of ICAM-1, CD44 and HLADR. The rate of CD154 expressing Th cells dropped at 12 months. For cT, no changes were seen for frequency or density. Immune reconstitution by oral CLAD was associated with modification of the pro-migratory and -inflammatory surface patterns of CAMs and CoSs in immune cell subsets. This observation pertains primarily to B cells, which are key cells underlying MS pathogenesis.