ITGAM sustains MAPK signaling and serves as an adverse prognostic factor and therapeutic target in acute myeloid leukemia.

Background: Acute myeloid leukemia (AML) is the second most frequently occurring type of leukemia in adults. Despite breakthroughs in genetics, the prognosis of AML patients remains dismal. The aim of this study is to find new therapeutic targets and diagnostic markers for AML and to explore their mechanisms of action. Methods: The expression patterns of integrin subunit alpha M (ITGAM) were investigated across different cell types using the Human Protein Atlas (HPA) database. The ITGAM levels across cancer types were analyzed using the Gene Expression Profiling Interactive Analysis (GEPIA) database. Prognostic correlations in AML individuals were evaluated using The Cancer Genome Atlas (TGCA) database. ITGAM-associated functions were evaluated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The AML cells were transfected with short-hairpin RNA targeting ITGAM or a control, and subsequently subjected to analysis in order to ascertain the impact of ITGAM on proliferation and apoptosis. Results: The expression of ITGAM was significantly higher in the AML patient samples compared to the control samples. High ITGAM expression was significantly associated with poor overall survival (OS). The knockdown of ITGAM in the AML cells resulted in a decrease in proliferation and an increase in apoptosis. This was accompanied by cell cycle arrest at the G1 phase and a downregulation of protein production for cyclin D1, cyclin E1, cyclin-dependent kinase 2 (CDK2), and cyclin-dependent kinase 4 (CDK4). A pathway analysis and a western blot analysis revealed that ITGAM positively regulated mitogen-activated protein kinase (MAPK) signaling by silencing attenuated p38 MAPK (P38), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) phosphorylation, while the total protein levels remained unchanged. Conclusions: ITGAM can serve as a potential prognostic biomarker and therapeutic target for AML. ITGAM production was elevated in AML and indicated poor survival. Silencing ITGAM suppressed AML cell viability and induced apoptosis by blocking cell cycle progression, likely by impeding the activation of the MAPK pathway. Further investigations that directly target the ITGAM-MAPK axis may offer novel strategies for mitigating AML pathogenesis and overcoming chemotherapy resistance.

Gene Regulation of Neutrophils Mediated Liver and Lung Injury through NETosis in Acute Pancreatitis.

Acute pancreatitis (AP) is one of the most common gastrointestinal emergencies, often resulting in self-digestion, edema, hemorrhage, and even necrosis of pancreatic tissue. When AP progresses to severe acute pancreatitis (SAP), it often causes multi-organ damage, leading to a high mortality rate. However, the molecular mechanisms underlying SAP-mediated organ damage remain unclear. This study aims to systematically mine SAP data from public databases and combine experimental validation to identify key molecules involved in multi-organ damage caused by SAP. Retrieve transcriptomic data of mice pancreatic tissue for AP, lung and liver tissue for SAP, and corresponding normal tissue from the Gene Expression Omnibus (GEO) database. Conduct gene differential analysis using Limma and DEseq2 methods. Perform enrichment analysis using the clusterProfiler package in R software. Score immune cells and immune status in various organs using single-sample gene set enrichment analysis (ssGSEA). Evaluate mRNA expression levels of core genes using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Validate serum amylase, TNF-α, IL-1ß, and IL-6 levels in peripheral blood using enzyme-linked immunosorbent assay (ELISA), and detect the formation of neutrophil extracellular traps (NETs) in mice pancreatic, liver, and lung tissues using immunofluorescence. Differential analysis reveals that 46 genes exhibit expression dysregulation in mice pancreatic tissue for AP, liver and lung tissue for SAP, as well as peripheral blood in humans. Functional enrichment analysis indicates that these genes are primarily associated with neutrophil-related biological processes. ROC curve analysis indicates that 12 neutrophil-related genes have diagnostic potential for SAP. Immune infiltration analysis reveals high neutrophil infiltration in various organs affected by SAP. Single-cell sequencing analysis shows that these genes are predominantly expressed in neutrophils and macrophages. FPR1, ITGAM, and C5AR1 are identified as key genes involved in the formation of NETs and activation of neutrophils. qPCR and IHC results demonstrate upregulation of FPR1, ITGAM, and C5AR1 expression in pancreatic, liver, and lung tissues of mice with SAP. Immunofluorescence staining shows increased levels of neutrophils and NETs in SAP mice. Inhibition of NETs formation can alleviate the severity of SAP as well as the levels of inflammation in the liver and lung tissues. This study identified key genes involved in the formation of NETs, namely FPR1, ITGAM, and C5AR1, which are upregulated during multi-organ damage in SAP. Inhibition of NETs release effectively reduces the systemic inflammatory response and liver-lung damage in SAP. This research provides new therapeutic targets for the multi-organ damage associated with SAP.

ITGAM-macrophage modulation as a potential strategy for treating neutrophilic Asthma: insights from bioinformatics analysis and in vivo experiments.

Identification of molecular biomarkers associated with neutrophilic asthma (NA) phenotype may inform the discovery of novel pathobiological mechanisms and the development of diagnostic markers. Three mRNA transcriptome datasets extracted from induced sputum of asthma patients with various inflammatory types were used to screen for macrophage-related molecular mechanisms and targets in NA. Furthermore, the predicted targets were also validated on an independent dataset (N = 3) and animal model (N = 5). A significant increase in total cells, neutrophils and macrophages was observed in bronchoalveolar lavage (BAL) fluid of NA mice induced by ovalbumin/freund's adjuvant, complete (OVA/CFA). And we also found elevated levels of neutrophil and macrophage infiltration in NA subtype in external datasets. NA mice had increased secretion of IgE, IL-1ß, TNF-α and IL-6 in serum and BAL fluid. MPO, an enzyme present in neutrophils, was also highly expressed in NA mice. Then, weighted gene co-expression network analysis (WGCNA) identified 684 targets with the strongest correlation with NA, and we obtained 609 macrophage-related specific differentially expressed genes (DEGs) in NA by integrating macrophage-related genes. The top 10 genes with high degree values were obtained and their mRNA levels and diagnostic performance were then determined by RT-qPCR and receiver operator characteristic (ROC) analysis. Statistically significant correlations were found between macrophages and all key targets, with the strongest correlation between ITGAM and macrophages in NA. Double-Immunofluorescence staining further confirmed the co-localization of ITGAM and F4/80 in NA. ITGAM was identified as a critical target to distinguish NA from healthy/non-NA individuals, which may provide a novel avenue to further uncover the mechanisms and therapy of NA.

NFIL3 aggravates human coronary artery endothelial cell injury by promoting ITGAM transcription in Kawasaki disease.

OBJECTIVE: High expression of nuclear factor interleukin-3 (NFIL3) and integrin Alpha M (ITGAM) was found in serum samples from Kawasaki disease (KD) patients through bioinformatics analysis. Hence, this study aimed to explore the biological functions of NFIL3 and ITGAM in KD serum-stimulated human coronary artery endothelial cells (HCAECs). METHODS: The differentially-expressed genes in KD were analyzed through bioinformatics analysis. Serum samples were obtained from 18 KD patients and 18 healthy volunteers, followed by detection of NFIL3 and ITGAM levels in KD serum. After HCAECs were transfected with sh-NFIL3, sh-ITGAM, or sh-NFIL3 + oe-ITGAM and underwent 24-h KD serum stimulation, cell viability and apoptosis and the levels of inflammation-related factors were measured. The binding between NFIL3 and ITGAM was validated by dual-luciferase and chromatin immunoprecipitation (ChIP) assays. RESULTS: NFIL3 and ITGAM were up-regulated in serum from KD patients and KD serum-stimulated HCAECs. Down-regulation of NFIL3 or ITGAM inhibited KD serum-induced cell apoptosis and inflammatory response of HCAECs and promoted cell viability. Mechanistically, NFIL3 promoted ITGAM transcription level. Up-regulation of ITGAM reversed the improvement of NFIL3 down-regulation on KD serum-induced HCAEC injury. CONCLUSION: NFIL3 aggravated KD serum-induced HCAEC injury by promoting ITGAM transcription, which provided new insights into the treatment of KD.

Bioinformatics analysis and identification of hub genes of neutrophils in Kawasaki disease: a pivotal study.

BACKGROUND: Kawasaki disease (KD) is considered the main contributor to acquired heart diseases in developed countries. However, the precise pathogenesis of KD remains unclear. Neutrophils play roles in KD. This study aimed to select hub genes in neutrophils in acute KD. METHODS: mRNA microarray of neutrophils from four acute KD patients and three healthy controls was performed to screen differentially expressed mRNAs (DE-mRNAs). DE-mRNAs were analyzed and predicted by Gene Ontology (GO), Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways, and protein-protein interaction networks. Real time-PCR was finally conducted to confirm the reliability and validity of the expression level of DE-mRNAs from blood samples of healthy controls and KD patients in both acute and convalescent stage. RESULTS: A total of 1950 DE-mRNAs including 1287 upregulated and 663 downregulated mRNAs were identified. GO and KEGG analyses revealed the DE-mRNAs were mainly enriched in the regulation of transcription from RNA polymerase II promoter, apoptotic process, intracellular signal transduction, protein phosphorylation, protein transport, metabolic pathways, carbon metabolism, lysosome, apoptosis, pyrimidine metabolism, alzheimer disease, prion disease, sphingolipid metabolism, huntington disease, glucagon signaling pathway, non-alcoholic fatty liver disease, pyruvate metabolism, sphingolipid signaling pathway, and peroxisome. Twenty hub DE-mRNAs were selected including GAPDH, GNB2L1, PTPRC, GART, HIST2H2AC, ACTG1, H2AFX, CREB1, ATP5A1, ENO1, RAC2, PKM, BCL2L1, ATP5B, MRPL13, SDHA, TLR4, RUVBL2, TXNRD1, and ITGAM. The real-time PCR results showed that BCL2L1 and ITGAM mRNA were upregulated in acute KD and were normalized in the convalescent stage. CONCLUSIONS: These findings may improve our understanding of neutrophils in KD. Key Points • Neutrophilic BCL2L1 and ITGAM mRNA were first reported to be correlated with the pathogenic mechanism of KD.

ITGAM rs1143679 Variant in Systemic Lupus Erythematosus Is Associated with Increased Serum Calcification Propensity.

OBJECTIVES: CD11B/ITGAM (Integrin Subunit α M) mediates the adhesion of monocytes, macrophages, and granulocytes and promotes the phagocytosis of complement-coated particles. Variants of the ITGAM gene are candidates for genetic susceptibility to systemic lupus erythematosus (SLE). SNP rs1143679 (R77H) of CD11B particularly increases the risk of developing SLE. Deficiency of CD11B is linked to premature extra-osseous calcification, as seen in the cartilage of animals with osteoarthritis. Serum calcification propensity measured by the T50 test is a surrogate marker for systemic calcification and reflects increased cardiovascular (CV) risk. We aimed to assess whether the CD11B R77H gene variant is associated with a higher serum calcification propensity (i.e., a lower T50 value) in SLE patients compared to the wild-type allele (WT). METHODS: Cross-sectional study incorporating adults with SLE genotyped for the CD11B variant R77H and assessed for serum calcification propensity with the T50 method. Participants were included in a multicenter trans-disciplinary cohort and fulfilled the 1997 revised American College of Rheumatology (ACR) criteria for SLE. We used descriptive statistics for comparing baseline characteristics and sequential T50 measurements in subjects with the R77H variant vs. WT CD11B. RESULTS: Of the 167 patients, 108 (65%) were G/G (WT), 53 (32%) were G/A heterozygous, and 6 (3%) were A/A homozygous for the R77H variant. A/A patients cumulated more ACR criteria upon inclusion (7 ± 2 vs. 5 ± 1 in G/G and G/A; p = 0.02). There were no differences between the groups in terms of global disease activity, kidney involvement, and chronic renal failure. Complement C3 levels were lower in A/A individuals compared to others (0.6 ± 0.08 vs. 0.9 ± 0.25 g/L; p = 0.02). Baseline T50 did not differ between the groups (A/A 278 ± 42' vs. 297 ± 50' in G/G and G/A; p = 0.28). Considering all sequential T50 test results, serum calcification propensity was significantly increased in A/A individuals compared to others (253 ± 50 vs. 290 ± 54; p = 0.008). CONCLUSIONS: SLE patients with homozygosity for the R77H variant and repeated T50 assessment displayed an increased serum calcification propensity (i.e., a lower T50) and lower C3 levels compared to heterozygous and WT CD11B, without differing with respect to global disease activity and kidney involvement. This suggests an increased CV risk in SLE patients homozygous for the R77H variant of CD11B.

Proteomic analysis of foot ulcer tissue reveals novel potential therapeutic targets of wound healing in diabetic foot ulcers.

Foot ulcers are a common complication of diabetes mellitus, which is associated with high morbidity and mortality among diabetic patients. The present study aims to investigate novel wound healing pathways in diabetic foot ulcers (DFU) through proteomics and a network pharmacology analysis. Tandem mass tag (TMT) labeled quantitative proteomics method was performed to evaluate the protein expression profile in wound tissues from healthy controls (HC) and DFU. Kyoto Encyclopedia of Genes (KEGG) and Genomes enrichment analysis (GO) was conducted based on differentially expressed proteins (DEPs) to discover the potential pathways associated with DFU. Western blot analysis was used to confirm the probable DFU-related targets. Proteomics analysis discovered 509 DEPs (248 upregulated and 261 downregulated proteins). Go and KEGG further evaluated the DEPs to discover the DFU-related pathways. According to network pharmacology study, three main targets (metalloproteinase 9 (MMP9), Fatty acid-binding protein 5 (FABP5), and integrin subunit alpha M (ITGAM)) play crucial roles in signaling pathways. Staphylococcus aureus infection and leukocyte transendothelial migration pathways significantly enriched in DFU. In addition, it was confirmed that three critical targets were elevated in diabetes mouse wound tissues. The study confirmed the presence of protein alterations in the wound-healing process of DFU mice and may provide fresh insights into the molecular mechanisms driving DFU.

Gene network landscape of mouse splenocytes reveals integrin complex as the A151 ODN-responsive hub molecule in the immune transcriptome.

Homeostatic restoration of an inflammatory response requires quenching of the immune system after pathogen threats vanish. A continued assault orchestrated by host defense results in tissue destruction or autoimmunity. A151 is the epitome of synthetic oligodeoxynucleotides (ODNs) that curb the immune response by a subset of white corpuscles through repetitive telomere-derived TTAGGG sequences. Currently, the genuine effect of A151 on the immune cell transcriptome remains unknown. Here, we leveraged an integrative approach where weighted gene co-expression network analysis (WGCNA), differential gene expression analysis, and gene set enrichment analysis (GSEA) of our in-house microarray datasets aided our understanding of how A151 ODN suppresses the immune response in mouse splenocytes. Our bioinformatics results, together with experimental validations, indicated that A151 ODN acts on components of integrin complexes, Itgam and Itga6, to interfere with immune cell adhesion and thereby suppresses the immune response in mice. Moreover, independent lines of evidence in this work converged on the observation that cell adhesion by integrin complexes serves as a focal point for cellular response to A151 ODN treatment in immune cells. Taken together, the outcome of this study sheds light on the molecular basis of immune suppression by a clinically useful DNA-based therapeutic agent.

Soluble ITGaM and ITGb2 Integrin Subunits Are Involved in Long-Term Pulmonary Complications after COVID-19 Infection.

(1) Introduction: The role of soluble integrins in post-COVID-19 complications is unclear, especially in long-term pulmonary lesions. The purpose of this study was to investigate the association between soluble ITGa2, ITGaM and ITGb2 integrin subunits and long COVID-19 pulmonary complications. (2) Methodology: Post-COVID-19 patients were enrolled. According to the evidence of persistent interstitial lung lesions on CT, patients were divided into a long-term pulmonary complications group (P(+)) and a control group without long-term pulmonary complications (P(-)). We randomly selected 80 patients for further investigation (40 subjects for each group). Levels of ITGa2, ITGaM and ITGb2 integrin subunits were determined by ELISA assay. (3) Results: The serum concentration of sITGaM and sITGb2 were significantly higher in the P(+) group (sITGaM 18.63 ng/mL [IQR 14.17-28.83] vs. 14.75 ng/mL [IQR 10.91-20] p = 0.01 and sITGb2 10.55 ng/mL [IQR 6.53-15.83] vs. 6.34 ng/mL [IQR 4.98-9.68] p = 0.002). We observed a statistically significant correlation between sITGaM and sITGb2 elevation in the P(+) group (R = 0.42; p = 0.01). Patients from the P(+) group had a lower (1.82 +/-0.84 G/L) lymphocyte level than the P(-)group (2.28 +/-0.79 G/L), p = 0.03. Furthermore, we observed an inverse correlation in the P(-) group between blood lymphocyte count and sITGb2 integrin subunit levels (R = -0.49 p = 0.01). (4) Conclusions: Elevated concentrations of sITGaM and sITGb2 were associated with long-term pulmonary complications in post-COVID-19 patients. Both sITGaM and sITGb2 may be promising biomarkers for predicting pulmonary complications and could be a potential target for therapeutic intervention in post-COVID-19 patients.

Identification of Key Diagnostic Markers and Immune Infiltration in Osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is a worldwide chronic disease of the articulating joints. An increasing body of data demonstrates the immune system's involvement in osteoarthritis. The molecular mechanisms of OA are still unclear. This study aimed to search for OA immunerelated hub genes and determine appropriate diagnostic markers to help the detection and treatment of the disease. METHODS: Gene expression data were downloaded from the GEO database. Firstly, we analyzed and identified the differentially expressed genes (DEGs) using R packages. Meanwhile, ssGSEA was used to determine the activation degree of immune-related genes (IRGs), and WGCNA analysis was applied to search for co-expressed gene modules associated with immune cells. Then, critical networks and hub genes were found in the PPI network. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway enrichment analyzed the biological functions of genes. The ability of the hub genes to differentiate OA from controls was assessed by the area under the ROC curve. A miRNA and transcription factor (TF) regulatory network was constructed according to their relationship with hub genes. Finally, the validation of hub genes was carried out by qPCR. RESULTS: In total, 353 DEGs were identified in OA patients compared with controls, including 222 upregulated and 131 downregulated genes. WGCNA successfully identified 34 main functional modules involved in the pathogenesis of OA. The most crucial functional module involved in OA included 89 genes. 19 immune-related genes were obtained by overlapping DEGs with the darkgrey module. The String database was constructed using the protein-protein interaction (PPI) network of 19 target genes, and 7 hub genes were identified by MCODE. ROC curve showed that 7 hub genes were potential biomarkers of OA. The expression levels of hub genes were validated by qPCR, and the results were consistent with those from bioinformatic analyses. CONCLUSION: Immune-related hub genes, including TYROBP, ITGAM, ITGB2, C1QC, MARCO, C1QB, and TLR8, may play critical roles in OA development. ITGAM had the highest correction on immune cells.

Association between complement gene polymorphisms and systemic lupus erythematosus: a systematic review and meta-analysis.

Complement dysfunction results in impaired ability in clearing apoptotic cell debris that may stimulate autoantibody production in systemic lupus erythematosus (SLE). Herein, we provided a comprehensive search to find and meta-analyze any complement gene polymorphisms associated with SLE. The ITGAM, C1q, and MBL gene polymorphisms were included in this meta-analysis to reveal the exact association with SLE risk. Electronic databases, including Scopus, PubMed, and Google Scholar, were searched to find studies investigating the ITGAM, C1q, and MBL gene polymorphisms and SLE risk in different populations. The pooled odds ratio (OR) and its corresponding 95% confidence interval (CI) were used to analyze the association between ITGAM, C1q, and MBL gene polymorphisms and susceptibility to SLE. According to inclusion criteria, a total of 24 studies, comprising 4 studies for C1QA rs292001, 5 studies for C1QA rs172378, 9 studies for ITGAM rs1143679, 8 studies for MBL rs1800450, 3 studies for MBL2 rs1800451, and 3 studies for MBL2 rs5030737, were included in the final meta-analysis. A significant positive association was found between rs1143679 and SLE risk, while rs1800451 significantly associated with decreased SLE susceptibility. In summary, ITGAM gene rs1143679 SNP and MBL gene rs1800451 SNP were positively and negatively associated with SLE risk, respectively.

Gingival crevicular fluid infiltrating CD14+ monocytes promote inflammation in periodontitis.

Periodontitis is a condition that occurs because of inflammation-mediated tissue degeneration. Many studies have been conducted to identify inflammatory molecules in periodontitis, but the well-defined role of cells from the immune system in the progression of periodontitis as well as in gingival tissue degeneration has not been appropriately established. The objective of the present study was to characterize the monocytes isolated from the gingival crevicular fluid (GCF) in patients with periodontitis. GCF was obtained from periodontitis patients and healthy controls. Cytokine levels of CCL2 were evaluated by ELISA in GCF samples. CD14+ monocytes were separated using magnetic sorting from GCF. RT-qPCR was performed to assess the gene expression. Cytometric bead array analysis was performed to analyze the levels of cytokines and chemokines in the secretome of cells. CD14+ monocytes from GCF secreted higher levels of CCL2 and showed elevated expression of genes responsible for monocyte migration. Additionally, upon lipopolysaccharide stimulation, these monocytes secreted higher levels of inflammatory cytokines and chemokines. This investigation aids in understanding the inflammatory microenvironment of periodontitis by characterizing GCF in terms of infiltrated CD14+ monocytes, cytokines, and molecules secreted by these monocytes, which are specific for cellular differentiation.

Human neutrophil antigens: Nature, clinical significance and detection.

Granulocytes are an essential part of both the innate and adaptive immune systems. Human neutrophil antigens (HNAs) are a family of epitopes that are located on glycoproteins that are mostly expressed on human granulocytes. Antibodies that recognize these epitopes have been associated with neutropenia, transfusion complications, haematopoietic stem cell transplant nonengraftment and renal transplant rejection. Currently, there are fourteen recognized HNA alleles across five antigen systems (HNA-1 through HNA-5), the molecular basis of which are located on the genes FCGR3B, CD177, SLC44A2, ITGAM and ITGAL, respectively. Elucidation of the associated genes has permitted the development of testing strategies for HNA typing and aided understanding of the associated epitopes. This review will outline the associated clinical conditions that require HNA investigation and how these are performed in specialized laboratories. Investigations provided are both reactive for patients with a variety of existing or suspected neutropenias and proactive in the testing of blood component donors in order to reduce the potential risk to patients who require transfusion.

Polymorphism of The Regulatory Region of the ITGAM Gene (-323G>A) as a Novel Predictor of a Poor Nutritional Status in Head and Neck Cancer Patients Subjected to Intensity-Modulated Radiation Therapy.

BACKGROUND: The most serious disturbance of the nutritional status is neoplastic cachexia. The main factor contributing to the development of cachexia is the ongoing inflammatory process. The gene associated with the development of the inflammatory response is ITGAM. Therefore, the aim of the study was to assess the relationship between a single nucleotide polymorphism (SNP)-323G>A of the ITGAM gene and the occurrence of nutritional disorders in patients undergoing radiotherapy (RT) due to head and neck cancers (HNC). METHODS: The study involved 71 patients with HNC treated with intensity-modulated radiotherapy (IMRT). SNP analysis of the ITGAM gene (-323G>A) was performed using commercial molecular probes and Real-Time PCR. RESULTS: The presence of the A allele of the ITGAM gene significantly (over 14-fold) reduced the risk of severe disturbances in nutritional status assessed according to the subjective global assessment (SGA) scale (odds ratio (OR) = 0.07; p = 0.0213). The GG genotype of this gene was associated with an over three-fold higher risk of shortened overall survival (OR = 3.01; p = 0.0376). CONCLUSIONS: Determination of the SNP (-323G>A) of the ITGAM gene may prove to be a useful marker in the assessment of the risk of nutritional disorders in patients with HNC undergoing RT.

Bidirectional Tumor-Promoting Activities of Macrophage Ezrin.

Ezrin links the cytoskeleton to cell surface integrins and plasma membrane receptors, contributing to the proliferative and metastatic potential of cancer cells. Elevated ezrin expression in several cancers is associated with poor outcomes. Tumor cell ezrin expression and function have been investigated in depth; however, its role in macrophages and other tumor microenvironment cells remains unexplored. Macrophages profoundly influence tumorigenesis, and here we explore ezrin's influence on tumor-promoting macrophage functions. Ezrin knockdown in THP-1 macrophages reveals its important contribution to adhesion to endothelial cells. Unexpectedly, ezrin is essential for the basal and breast cancer cell-stimulated THP-1 expression of ITGAM mRNA that encodes integrin CD11b, critical for cell adhesion. Ezrin skews the differentiation of THP-1 macrophages towards the pro-tumorigenic, M2 subtype, as shown by the reduced expression of FN1, IL10, and CCL22 mRNAs following ezrin knockdown. Additionally, macrophage ezrin contributes to the secretion of factors that stimulate tumor cell migration, invasion, and clonogenic growth. Lastly, THP-1 ezrin is critical for the expression of mRNAs encoding vascular endothelial growth factor (VEGF)-A and matrix metalloproteinase (MMP)-9, consistent with pro-tumorigenic function. Collectively, our results provide insight into ezrin's role in tumorigenesis, revealing a bidirectional interaction between tumor-associated macrophages and tumor cells, and suggest myeloid cell ezrin as a target for therapeutic intervention against cancer.

Association of ITGAM, TNFSF4, TNFAIP3 and STAT4 gene polymorphisms with risk of systemic lupus erythematosus in a North Indian population.

Several susceptibility genes have been associated with systemic lupus erythematosus (SLE) across different populations worldwide. However, data on association between genetic polymorphisms and SLE from Indian population is scarce. We aimed to replicate the association of single nucleotide polymorphisms (SNPs) in ITGAM, TNFSF4, TNFAIP3 and STAT4 genes with susceptibility to SLE in a North Indian population. Three hundred and ninety-four SLE patients and 583 unrelated healthy controls of the same ethnic background were enrolled. All samples were genotyped for SNPs in ITGAM (rs1143679), TNFSF4 (rs2205960), TNFAIP3 (rs5029939) and STAT4 (rs7574865) using TaqMan genotyping assay. At allele level, significant association with susceptibility to SLE was detected with polymorphisms in ITGAM (A vs. G, odds ratio (OR) = 1.73, 95% confidence interval (CI) = 1.30-2.30, p < 0.001), TNFSF4 (T vs. G, OR = 1.33, 95% CI = 1.08-1.64, p < 0.01), TNFAIP3 (G vs. C, OR = 1.91, 95% CI = 1.27-2.85, p < 0.01) and STAT4 (T vs. G, OR = 1.38, 95% CI = 1.13-1.69, p < 0.01). All four SNPs were associated with SLE under a dominant model with an OR of 1.47 (95% CI = 1.07-2.04, p < 0.05) for ITGAM, 1.30 (95% CI = 1.01-1.69, p < 0.05) for TNFSF4, 1.90 (95% CI = 1.25-2.90, p < 0.01) for TNFAIP3 and 1.38 (95% CI = 1.06-1.78, p < 0.05) for STAT4. Under a recessive model, significant association was found with ITGAM (OR = 4.87, 95% CI = 2.17-10.91, p < 0.001), TNFSF4 (OR = 1.84, 95% CI = 1.13-3.00, p < 0.05) and STAT4 (OR = 1.82, 95% CI = 1.19-2.77, p < 0.01). In conclusion, single nucleotide polymorphisms in ITGAM, TNFSF4, TNFAIP3 and STAT4 genes are associated with susceptibility to SLE in a North Indian population.

CD11b Activity Modulates Pathogenesis of Lupus Nephritis.

Lupus nephritis (LN) is a common complication of systemic lupus erythematosus (SLE) with unclear etiology and limited treatment options. Immune cell infiltration into the kidneys, a hallmark of LN, triggers tissue damage and proteinuria. CD11b, the α-chain of integrin receptor CD11b/CD18 (also known as αMß2, Mac-1, and CR3), is highly expressed on the surface of innate immune cells, including macrophages and neutrophils. Genetic variants in the human ITGAM gene, which encodes for CD11b, are strongly associated with susceptibility to SLE, LN, and other complications of SLE. CD11b modulates several key biological functions in innate immune cells, including cell adhesion, migration, and phagocytosis. CD11b also modulates other signaling pathways in these cells, such as the Toll-like receptor signaling pathways, that mediate generation of type I interferons, a key proinflammatory cytokine and circulating biomarker in SLE and LN patients. However, how variants in ITGAM gene contribute to disease pathogenesis has not been completely established. Here, we provide an overview of CD11b modulated mechanisms and the functional consequences of the genetic variants that can drive disease pathogenesis. We also present recent insights from studies after pharmacological activation of CD11b. These studies offer novel mechanisms for development of therapeutics for LN, SLE and other autoimmune diseases.

Vascular Endothelial Cell-Specific Connective Tissue Growth Factor (CTGF) Is Necessary for Development of Chronic Hypoxia-Induced Pulmonary Hypertension.

Chronic hypoxia frequently complicates the care of patients with interstitial lung disease, contributing to the development of pulmonary hypertension (PH), and premature death. Connective tissue growth factor (CTGF), a matricellular protein of the Cyr61/CTGF/Nov (CCN) family, is known to exacerbate vascular remodeling within the lung. We have previously demonstrated that vascular endothelial-cell specific down-regulation of CTGF is associated with protection against the development of PH associated with hypoxia, though the mechanism for this effect is unknown. In this study, we generated a transgenic mouse line in which the Ctgf gene was floxed and deleted in vascular endothelial cells that expressed Cre recombinase under the control of VE-Cadherin promoter (eCTGF KO mice). Lack of vascular endothelial-derived CTGF protected against the development of PH secondary to chronic hypoxia, as well as in another model of bleomycin-induced pulmonary hypertension. Importantly, attenuation of PH was associated with a decrease in infiltrating inflammatory cells expressing CD11b or integrin αM (ITGAM), a known adhesion receptor for CTGF, in the lungs of hypoxia-exposed eCTGF KO mice. Moreover, these pathological changes were associated with activation of-Rho GTPase family member-cell division control protein 42 homolog (Cdc42) signaling, known to be associated with alteration in endothelial barrier function. These data indicate that endothelial-specific deletion of CTGF results in protection against development of chronic-hypoxia induced PH. This protection is conferred by both a decrease in inflammatory cell recruitment to the lung, and a reduction in lung Cdc42 activity. Based on our studies, CTGF inhibitor treatment should be investigated in patients with PH associated with chronic hypoxia secondary to chronic lung disease.

The heteromeric transcription factor GABP activates the ITGAM/CD11b promoter and induces myeloid differentiation.

The heteromeric transcription factor GA-binding protein (GABP) consists of two subunits, the alpha subunit (GABPA) carrying the DNA-binding ETS domain, and the beta subunit (GABPB1) harbouring the transcriptional activation domain. GABP is involved in haematopoietic stem cell maintenance and differentiation of myeloid and lymphoid lineages in mice. To elucidate the molecular function of GABP in human haematopoiesis, the present study addressed effects of ectopic overexpression of GABP focussing on the myeloid compartment. Combined overexpression of GABPA and GABPB1 caused a proliferation block in cell lines and drastically reduced the colony-forming capacity of murine lineage-negative cells. Impaired proliferation resulted from perturbed cellular cycling and induction of myeloid differentiation shown by surface markers and myelomonocytic morphology of U937 cells. Depending on the dosage and functional integrity of GABP, ITGAM expression was induced. ITGAM encodes CD11b, the alpha subunit of integrin Mac-1, whose beta subunit, ITGB2/CD18, was already described to be regulated by GABP. Finally, Shield1-dependent proteotuning, luciferase reporter assays and chromatin immunoprecipitation showed that GABP activates the ITGAM/CD11b promoter via three binding sites close to the translational start site. In conclusion, the present study supports the crucial role of GABP in myeloid cell differentiation and identified ITGAM/CD11b as a novel GABP target gene.

The PRKAA1/AMPKα1 pathway triggers autophagy during CSF1-induced human monocyte differentiation and is a potential target in CMML.

Autophagy is induced during differentiation of human monocytes into macrophages that is mediated by CSF1/CSF-1/M-CSF (colony stimulating factor 1 [macrophage]). However, little is known about the molecular mechanisms that link CSF1 receptor engagement to the induction of autophagy. Here we show that the CAMKK2-PRKAA1-ULK1 pathway is required for CSF1-induced autophagy and human monocyte differentiation. We reveal that this pathway links P2RY6 to the induction of autophagy, and we decipher the signaling network that links the CSF1 receptor to P2RY6-mediated autophagy and monocyte differentiation. In addition, we show that the physiological P2RY6 ligand UDP and the specific P2RY6 agonist MRS2693 can restore normal monocyte differentiation through reinduction of autophagy in primary myeloid cells from some but not all chronic myelomonocytic leukemia (CMML) patients. Collectively, our findings highlight an essential role for PRKAA1-mediated autophagy during differentiation of human monocytes and pave the way for future therapeutic interventions for CMML.