The Microglial Transcriptome of Age-Associated Deep Subcortical White Matter Lesions Suggests a Neuroprotective Response to Blood-Brain Barrier Dysfunction.

Age-associated deep-subcortical white matter lesions (DSCLs) are an independent risk factor for dementia, displaying high levels of CD68+ microglia. This study aimed to characterize the transcriptomic profile of microglia in DSCLs and surrounding radiologically normal-appearing white matter (NAWM) compared to non-lesional control white matter. CD68+ microglia were isolated from white matter groups (n = 4 cases per group) from the Cognitive Function and Ageing Study neuropathology cohort using immuno-laser capture microdissection. Microarray gene expression profiling, but not RNA-sequencing, was found to be compatible with immuno-LCM-ed post-mortem material in the CFAS cohort and identified significantly differentially expressed genes (DEGs). Functional grouping and pathway analysis were assessed using the Database for Annotation Visualization and Integrated Discovery (DAVID) software, and immunohistochemistry was performed to validate gene expression changes at the protein level. Transcriptomic profiling of microglia in DSCLs compared to non-lesional control white matter identified 181 significant DEGs (93 upregulated and 88 downregulated). Functional clustering analysis in DAVID revealed dysregulation of haptoglobin-haemoglobin binding (Enrichment score 2.5, p = 0.017), confirmed using CD163 immunostaining, suggesting a neuroprotective microglial response to blood-brain barrier dysfunction in DSCLs. In NAWM versus control white matter, microglia exhibited 347 DEGs (209 upregulated, 138 downregulated), with significant dysregulation of protein de-ubiquitination (Enrichment score 5.14, p < 0.001), implying an inability to maintain protein homeostasis in NAWM that may contribute to lesion spread. These findings enhance understanding of microglial transcriptomic changes in ageing white matter pathology, highlighting a neuroprotective adaptation in DSCLs microglia and a potentially lesion-promoting phenotype in NAWM microglia.

Single-cell RNA sequencing analysis of vestibular schwannoma reveals functionally distinct macrophage subsets.

BACKGROUND: Vestibular schwannomas (VSs) remain a challenge due to their anatomical location and propensity to growth. Macrophages are present in VS but their roles in VS pathogenesis remains unknown. OBJECTIVES: The objective was to assess phenotypic and functional profile of macrophages in VS with single-cell RNA sequencing (scRNAseq). METHODS: scRNAseq was carried out in three VS samples to examine characteristics of macrophages in the tumour. RT-qPCR was carried out on 10 VS samples for CD14, CD68 and CD163 and a panel of macrophage-associated molecules. RESULTS: scRNAseq revealed macrophages to be a major constituent of VS microenvironment with three distinct subclusters based on gene expression. The subclusters were also defined by expression of CD163, CD68 and IL-1ß. AREG and PLAUR were expressed in the CD68+CD163+IL-1ß+ subcluster, PLCG2 and NCKAP5 were expressed in CD68+CD163+IL-1ß- subcluster and AUTS2 and SPP1 were expressed in the CD68+CD163-IL-1ß+ subcluster. RT-qPCR showed expression of several macrophage markers in VS of which CD14, ALOX15, Interleukin-1ß, INHBA and Colony Stimulating Factor-1R were found to have a high correlation with tumour volume. CONCLUSIONS: Macrophages form an important component of VS stroma. scRNAseq reveals three distinct subsets of macrophages in the VS tissue which may have differing roles in the pathogenesis of VS.

The glycoimmune checkpoint receptor Siglec-7 interacts with T-cell ligands and regulates T-cell activation.

Siglec-7 (sialic acid-binding immunoglobulin-like lectin 7) is a glycan-binding immune receptor that is emerging as a significant target of interest for cancer immunotherapy. The physiological ligands that bind Siglec-7, however, remain incompletely defined. In this study, we characterized the expression of Siglec-7 ligands on peripheral immune cell subsets and assessed whether Siglec-7 functionally regulates interactions between immune cells. We found that disialyl core 1 O-glycans are the major immune ligands for Siglec-7 and that these ligands are particularly highly expressed on naïve T-cells. Densely glycosylated sialomucins are the primary carriers of these glycans, in particular a glycoform of the cell-surface marker CD43. Biosynthesis of Siglec-7-binding glycans is dynamically controlled on different immune cell subsets through a genetic circuit involving the glycosyltransferase GCNT1. Siglec-7 blockade was found to increase activation of both primary T-cells and antigen-presenting dendritic cells in vitro, indicating that Siglec-7 binds T-cell glycans to regulate intraimmune signaling. Finally, we present evidence that Siglec-7 directly activates signaling pathways in T-cells, suggesting a new biological function for this receptor. These studies conclusively demonstrate the existence of a novel Siglec-7-mediated signaling axis that physiologically regulates T-cell activity. Going forward, our findings have significant implications for the design and implementation of therapies targeting immunoregulatory Siglec receptors.

Ectopic expression of murine CD163 enables cell-culture isolation of lactate dehydrogenase-elevating virus 63 years after its discovery.

IMPORTANCE: Mouse models of viral infection play an especially large role in virology. In 1960, a mouse virus, lactate dehydrogenase-elevating virus (LDV), was discovered and found to have the peculiar ability to evade clearance by the immune system, enabling it to persistently infect an individual mouse for its entire lifespan without causing overt disease. However, researchers were unable to grow LDV in culture, ultimately resulting in the demise of this system as a model of failed immunity. We solve this problem by identifying the cell-surface molecule CD163 as the critical missing component in cell-culture systems, enabling the growth of LDV in immortalized cell lines for the first time. This advance creates abundant opportunities for further characterizing LDV in order to study both failed immunity and the family of viruses to which LDV belongs, Arteriviridae (aka, arteriviruses).

[Effect of catgut implantation on macrophage CD68, tumor necrosis factor-α and interleukin-1ß in "Zusanli" (ST 36) region of rats].

OBJECTIVE: To observe the expression of local macrophages and related cytokines tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) after catgut implantation in "Zusanli"(ST 36) in rats, so as to explore its underlying mechanisms in inducing therapeutic effect. METHODS: A total of 110 male SD rats were randomly divided into blank control group (n=10), catgut embedding (CE) group (n=50), and sham CE group (n=50). The CE and sham CE groups were randomly divided into 8 h, 3 d, 7 d, 14 d and 21 d subgroups after the intervention (n=10 in each time point group). Rats of the CE group were uniformly subjected into catgut embedding at ST36 once, and those of the sham CE group received embedding needle puncture at ST36 without catgut retention, and the blank control group was only grasped and fixed without other treatments. Tissues from the ST36 area in each group were collected at the corresponding time points, and the expression of CD68 in macrophages in the acupoint area was detected by immunofluorescence, the contents of TNF-α and IL-1ß in the acupoint area were detected by ELISA. RESULTS: Following catgut embedment at ST36, the contents of TNF-α and IL-1ß, and macrophage CD68 expression level began to increase at 8 h, peaked at 3 d, and then gradually decreased at 7, 14, and 21 d, being still higher in the CE group than in the blank control group at 21 d (P<0.05). Compared with the blank control group, the contents of TNF-α and IL-1ß, and macrophage CD68 expression were significantly increased at 8 h, and 3, 7, 14 and 21 d in the CE group (P<0.05). Following sham CE at ST36, the content of TNF-α at 8 h and 3 d, IL-1ß at 8 h and 3, 7 and 14 d, and expression of CD68 at 8 h were significantly increased in comparison with the blank control group (P<0.05). Comparison between the CE and sham CE groups showed that the contents of IL-1ß at 3, 7, 14 and 21 d, and contents of TNF-α，CD68 expression at 8 h, and 3, 7, 14 and 21 d were significantly higher in the CE group than in the sham CE group (P<0.05). CONCLUSION: Catgut embedding at ST36 can induce an increase levels of inflammatory cytokines TNF-α, IL-1ß and macrophage CD68 in the local microenvironment in rats, which may contribute to its functions in initiating therapeutic effect.

Identification of New Compounds against PRRSV Infection by Directly Targeting CD163.

The porcine reproductive and respiratory syndrome viruses (PRRSV) led to a global panzootic and huge economical losses to the pork industry. PRRSV targets the scavenger receptor CD163 for productive infection. However, currently no effective treatment is available to control the spread of this disease. Using bimolecular fluorescence complementation (BiFC) assays, we screened a set of small molecules potentially targeting the scavenger receptor cysteine-rich domain 5 (SRCR5) of CD163. We found that the assay examining protein-protein interactions (PPI) between PRRSV glycoprotein 4 (GP4) and the CD163-SRCR5 domain mainly identifies compounds that potently inhibit PRRSV infection, while examining the PPI between PRRSV-GP2a and the SRCR5 domain maximized the identification of positive compounds, including additional ones with various antiviral capabilities. These positive compounds significantly inhibited both types 1 and 2 PRRSV infection of porcine alveolar macrophages. We confirmed that the highly active compounds physically bind to the CD163-SRCR5 protein, with dissociation constant (KD) values ranging from 28 to 39 µM. Structure-activity-relationship (SAR) analysis revealed that although both the 3-(morpholinosulfonyl)anilino and benzenesulfonamide moieties in these compounds are critical for the potency to inhibit PRRSV infection, the morpholinosulfonyl group can be replaced by chlorine substituents without significant loss of antiviral potency. Our study established a system for throughput screening of natural or synthetic compounds highly effective on blocking of PRRSV infection and shed light on further SAR modification of these compounds. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the swine industry worldwide. Current vaccines cannot provide cross protection against different strains, and there are no effective treatments available to hamper the spread of this disease. In this study, we identified a group of new small molecules that can inhibit the PRRSV interaction with its specific receptor CD163 and dramatically block the infection of both types 1 and type 2 PRRSVs to host cells. We also demonstrated the physical association of these compounds with the SRCR5 domain of CD163. In addition, molecular docking and structure-activity relationship analyses provided new insights for the CD163/PRRSV glycoprotein interaction and further improvement of these compounds against PRRSV infection.

Case-only design identifies interactions of genetic risk variants at SIGLEC5 and PLG with the lncRNA CTD-2353F22.1 implying the importance of periodontal wound healing for disease aetiology.

AIM: The basis of phenotypic variation of periodontitis is genetic variability. Disease relevant effects of individual risk alleles are considered to result from genetic interactions. We investigated gene × gene (G×G) interactions of suggestive periodontitis susceptibility alleles. MATERIALS AND METHODS: We used the case-only design and investigated single-nucleotide polymorphism (SNPs) that showed associations in our recent genome-wide association study (GWAS) and GWAS meta-analysis with p < 5 × 10-6 . CRISPR-dCas9 gene activation followed by RNA-sequencing and gene-set enrichment analyses elucidated differentially expressed genes and gene networks. With the databases of SNPInspector and Transfac professional, luciferase reporter gene assays and antibody electrophoretic mobility shift experiments, we analysed allele-specific effects on transcription factor binding. RESULTS: SNPs at the genes sialic acid binding Ig-like lectin 5 (SIGLEC5) and plasminogen (PLG) showed G×G interactions with rs1122900 at the long non-coding RNA (lncRNA) CTD-2353F22. Associated chromatin cis-activated CTD-2353F22.1 6.5-fold (p = .003), indicating CTD-2353F22.1 as target gene of this interaction. CTD-2353F22.1 regulated GADD45A (padj < 4.9 × 10-11 , log2 fold change (FC) = -0.55), THBS1, SERPINE1 and Tissue Factor F3 (padj < 5 × 10-7 , log2 FC ≥ -0.35) and the gene set "angiogenesis" (area under the curve = 0.71, padj  = 8.2 × 10-5 ). rs1122900 effect C-allele decreased reporter gene activity (5.5-fold, p = .0003) and PRDM14 binding (76%). CONCLUSIONS: CTD-2353F22.1 mediates interaction of SIGLEC5 and PLG, together with genes that function in periodontal wound healing.

Circulating Soluble CD163, Associations With Cardiovascular Outcomes and Mortality, and Identification of Genetic Variants in Older Individuals: The Cardiovascular Health Study.

Background Monocytes/macrophages participate in cardiovascular disease. CD163 (cluster of differentiation 163) is a monocyte/macrophage receptor, and the shed sCD163 (soluble CD163) reflects monocyte/macrophage activation. We examined the association of sCD163 with incident cardiovascular disease events and performed a genome-wide association study to identify sCD163-associated variants. Methods and Results We measured plasma sCD163 in 5214 adults (aged ≥65 years, 58.7% women, 16.2% Black) of the CHS (Cardiovascular Health Study). We used Cox regression models (associations of sCD163 with incident events and mortality); median follow-up was 26 years. Genome-wide association study analyses were stratified on race. Adjusted for age, sex, and race and ethnicity, sCD163 levels were associated with all-cause mortality (hazard ratio [HR], 1.08 [95% CI, 1.04-1.12] per SD increase), cardiovascular disease mortality (HR, 1.15 [95% CI, 1.09-1.21]), incident coronary heart disease (HR, 1.10 [95% CI, 1.04-1.16]), and incident heart failure (HR, 1.18 [95% CI, 1.12-1.25]). When further adjusted (eg, cardiovascular disease risk factors), only incident coronary heart disease lost significance. In European American individuals, genome-wide association studies identified 38 variants on chromosome 2 near MGAT5 (top result rs62165726, P=3.3×10-18),19 variants near chromosome 17 gene ASGR1 (rs55714927, P=1.5×10-14), and 18 variants near chromosome 11 gene ST3GAL4. These regions replicated in the European ancestry ADDITION-PRO cohort, a longitudinal cohort study nested in the Danish arm of the Anglo-Danish-Dutch study of Intensive Treatment Intensive Treatment In peOple with screeNdetcted Diabetes in Primary Care. In Black individuals, we identified 9 variants on chromosome 6 (rs3129781 P=7.1×10-9) in the HLA region, and 3 variants (rs115391969 P=4.3×10-8) near the chromosome 16 gene MYLK3. Conclusions Monocyte function, as measured by sCD163, may be predictive of overall and cardiovascular-specific mortality and incident heart failure.

Elevated trophoblastic Siglec6 contributes to the impairment of vascular endothelial cell functions by downregulating Wnt6/ß-catenin signaling in preeclampsia.

Preeclampsia (PE), a systemic vascular disorder, is the leading cause of maternal and perinatal morbidity and mortality, and its pathogenesis has yet to be fully elucidated. Siglec6, a transmembrane protein, is highly expressed in human placental trophoblasts, and previous studies have shown that Siglec6 overexpression correlates with PE, but the role of Siglec6 during PE progression is unknown. Here, we demonstrated that the mRNA and protein expression levels of Siglec6 were upregulated in early-onset PE placentas compared with uncomplicated pregnancies, and Siglec6 was primarily located in syncytiotrophoblasts (STBs) and extravillous trophoblasts (EVTs). Moreover, our results showed that chemical reagent-induced HIF-1α accumulation promoted the mRNA and protein levels of Siglec6 in HTR8/SVneo and BeWo cells. Although Siglec6 overexpression did not affect HTR8/SVneo cell proliferation, migration, and invasion, the conditional medium derived from the Siglec6 overexpressed HTR8/SVneo cells (Siglec6-OE-CM) significantly impaired the proliferation, migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs). Subsequently, the transcriptome sequencing results revealed that Siglec6 overexpression led to the downregulation of Wnt6 in HTR8/SVneo cells, which was further confirmed by qPCR and ELISA. Recombinant human Wnt6 reversed Siglec6-OE-CM-mediated suppression of HUVEC functions by reactivating the Wnt/ß-catenin signaling pathway. Altogether, our study found that elevated trophoblastic Siglec6 contributed to the impairment of vascular endothelial cell functions by downregulating Wnt6/ß-catenin signaling.

CD163-Expressing Porcine Macrophages Support NADC30-like and NADC34-like PRRSV Infections.

Porcine reproductive and respiratory syndrome virus (PRRSV) has a strict cell tropism. In addition to the primary alveolar macrophages, PRRSV is strictly cytotropic to African green monkey kidney cells, such as MARC-145 cells; however, MARC-145 cells are not infected by most NADC30-like and NADC34-like PRRSV strains. The essential scavenger receptor CD163 has been proved to mediate productive infection of PRRSV in various non-permissive cell lines. In this study, we systematically tested the porcine CD163 stably expressing 3D4/21 cells for infections with various PRRSV strains. The results showed that the porcine CD163-expressing macrophages support the infections of PRRSV2 of lineages 1, 5, and 8, as evidenced by Western blotting, immunofluorescence assay, quantitative PCR, and virus titration assay. Considering the current prevalence of NADC30-like and NADC34-like PRRSV2 of lineage 1 in China, the CD163-expressing macrophages are very useful for PRRSV research and disease management.

Identification of CD163 regions that are required for porcine reproductive and respiratory syndrome virus (PRRSV) infection but not for binding to viral envelope glycoproteins.

CD163, a receptor for porcine reproductive and respiratory syndrome virus (PRRSV), possesses nine scavenger receptor cysteine-rich (SRCR) and two proline-serine-threonine (PST) domains. To identify CD163 regions involved in PRRSV infection, CD163 mutants were generated. Infection experiments showed resistance to infection following deletion of the SRCR4/5 interdomain or the Exon 13 that encodes a portion of PSTII. The mutation of a pentapeptide domain in SRCR5 and SRCR7 also conferred resistance. Mutant CD163 proteins that resisted infection retained the ability to interact with GP2, GP3, GP4 and GP5 viral glycoproteins. The contribution of multiple domains to infection but not to the binding of viral glycoproteins suggests that the envelope proteins may form multiple interactions with CD163, or that receptor regions important for infection have other cellular binding partners required for PRRSV infection. Finally, we mapped the localization the anti-CD163 2A10 antibody epitope.

Low IL7R Expression at Diagnosis Predicted Relapse in Adult Acute Myeloid Leukemia Patients With t(8;21).

Background: Acute myeloid leukemia (AML) with t(8;21) needs to be further stratified. In addition to leukemia cells, immune cells in tumor microenvironment participate in tumor initiation, growth and progression. Interleukins (ILs)/interleukin receptors (ILRs) interaction plays important roles in the antitumor immune response. IL7R is reported to be relevant to prognosis in solid tumor and acute lymphoblastic leukemia. However, the prognostic significance of IL7R in t(8;21) AML remains to be clarified. Methods: Bone marrows collected from 156 newly diagnosed t(8;21) AML patients were used for testing IL7R transcript level by TaqMan-based real-time quantitative PCR (RQ-PCR), and RNAseq were performed in 15 of them. Moreover, IL7R expression at diagnosis were measured by RQ-PCR and flow cytometry (FCM) simultaneously in other 13 t(8;21) AML patients. Results: t(8;21) AML patients had varied IL7R transcript levels and were categorized into low-expression (IL7R-L) and high-expression (IL7R-H) groups; IL7R-L was significantly associated with a lower relapse-free survival (RFS) rate (P=0.0027) and KITD816/D820 mutation (P=0.0010). Furthermore, IL7R-L was associated with a lower RFS rate in KITD816/D820 group (P=0.013) and IL7R-H/KITD816/D820 patients had similar RFS to KITN822/e8/WT patients (P=0.35). GO analysis enrichment showed that down-regulated genes were predominantly involved in the regulation of T cell and leukocyte activation, proliferation and differentiation in IL7R-L group. IL7R-L had significantly lower levels of Granzymes A/B, CCR7, CD28 and CD27 than IL7R-H group (all P<0.05). FCM analysis showed IL7R protein was primarily expressed in CD4+ T and CD8+ T cell subset. A significant association was found between the transcript level of IL7R and the percentage of CD8+ T cells in nucleated cells (P=0.015) but not CD4+ T cells (P=0.47). Conclusion: Low IL7R transcript level of bone marrow at diagnosis predicted relapse in t(8;21) AML, which might be caused by the difference in the amount, status and function of T cells.

Impact of thymidine phosphorylase and CD163 expression on prognosis in stage II colorectal cancer.

BACKGROUND: Tumor-associated macrophages (TAM) are known to facilitate colorectal cancer (CRC) growth. High macrophage infiltration in thymidine phosphorylase (TYMP) expressing CRC may correspond to poor prognosis. The prognostic impact of the expression CD163, a receptor associated with TAM, and TYMP in stroma, respectively, tumor tissue is not yet established. The aim of this study was to identify the potential associations between TYMP and CD163 expression levels and relapse-free survival (RFS) of patients with stage II CRC, and if microdissection is of importance. METHODS: Stage II CRC patients, radically resected with relapse (n = 104), were matched to patients with a 5-year relapse-free follow-up (n = 206). Gene expression of TYMP and CD163 was analyzed in snap-frozen tumor tissues and in microdissected formalin-fixed tumor tissues separated into tumor epithelium and stroma. RESULTS: TYMP expression was high in poorly differentiated tumors, right-sided CRC, and tumors with high microsatellite instability CD163-expressing macrophages near tumor epithelial cells had high expression in poorly differentiated and T4 tumors. High TYMP expression in tumor epithelial cells was in the multivariate analyses associated with shorter relapse-free survival (hazard ratio 1.66; 95% confidence interval: 1.09-2.56; p < 0.05). CONCLUSIONS: TYMP expression in tumor epithelial cells was associated with RFS and emphasizes the need for tissue microdissection. Additional studies are needed to establish whether TYMP and CD163 could add clinically relevant information to identify high-risk stage II patients that could benefit from adjuvant chemotherapy.

Mutations within scavenger receptor cysteine-rich (SRCR) protein domain 5 of porcine CD163 involved in infection with porcine reproductive and respiratory syndrome virus (PRRS).

CD163, a macrophage-specific membrane scavenger receptor, serves as a cellular entry receptor for porcine reproductive and respiratory syndrome virus (PRRSV). The removal of scavenger receptor cysteine-rich (SRCR) domain 5 (SRCR5) of CD163 is sufficient to make transfected cells or genetically modified pigs resistant to PRRSV-1 and PRRSV-2 genotypes, and substitution of SRCR5 with SRCR8 from human CD163-like protein (hCD163L1) confers resistance to PRRSV-1 but not PRRSV-2 isolates. However, the specific regions within the SRCR5 polypeptide involved in PRRSV infection remain largely unknown. In this report, we performed mutational studies in order to identify which regions or amino acid sequences in the SRCR5 domain are critical for PRRSV infection. The approach used in this study was to make proline-arginine (PR) insertions along the SRCR5 polypeptide. Constructs were transfected into HEK293T cells, and then evaluated for infection with PRRSV-2 or PRRSV-1. For PRRSV-2, four PR insertions located after amino acids 8 (PR-9), 47 (PR-48), 54 (PR-55), and 99 (PR-100) had the greatest impact on infection. For PRRSV-1, insertions after amino acids 57 (PR-58) and 99 (PR-100) were critical. Computer simulations based on the crystal structure of SRCR5 showed that the mutations that affected infection localized to a similar region on the surface of the 3-D structure. Specifically, we found two surface patches that are essential for PRRSV infection. PR-58 and PR-55, which were separated by only three amino acids, had reciprocal effects on PRRSV-1 and PRRSV-2. Substitution of Glu-58 with Lys-58 reduced PRRSV-1 infection without affecting PRRSV-2, which partially explains the resistance to PRRSV-1 caused by the SRCR5 replacement with the homolog human SRCR8 previously observed. Finally, resistance to infection was observed following the disruption of any of the four conserved disulfide bonds within SRCR5. In summary, the results confirm that there are distinct differences between PRRSV-1 and PRRSV-2 on recognition of CD163; however, all mutations that affect infection locate on a similar region on the same face of SRCR5.

Functional and Phenotypic Characterization of Siglec-6 on Human Mast Cells.

Mast cells are tissue-resident cells that contribute to allergic diseases, among others, due to excessive or inappropriate cellular activation and degranulation. Therapeutic approaches to modulate mast cell activation are urgently needed. Siglec-6 is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing receptor selectively expressed by mast cells, making it a promising target for therapeutic intervention. However, the effects of its engagement on mast cells are poorly defined. Siglec-6 expression and endocytosis on primary human mast cells and mast cell lines were assessed by flow cytometry. SIGLEC6 mRNA expression was examined by single-cell RNAseq in esophageal tissue biopsy samples. The ability of Siglec-6 engagement or co-engagement to prevent primary mast cell activation was determined based on assessments of mediator and cytokine secretion and degranulation markers. Siglec-6 was highly expressed by all mast cells examined, and the SIGLEC6 transcript was restricted to mast cells in esophageal biopsy samples. Siglec-6 endocytosis occurred with delayed kinetics relative to the related receptor Siglec-8. Co-crosslinking of Siglec-6 with FcεRIα enhanced the inhibition of mast cell activation and diminished downstream ERK1/2 and p38 phosphorylation. The selective, stable expression and potent inhibitory capacity of Siglec-6 on human mast cells are favorable for its use as a therapeutic target in mast cell-driven diseases.

ACE2 protein expression in lung tissues of severe COVID-19 infection.

Angiotensin-converting enzyme 2 (ACE2) is a key host protein by which severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) enters and multiplies within cells. The level of ACE2 expression in the lung is hypothesised to correlate with an increased risk of severe infection and complications in COrona VIrus Disease 2019 (COVID-19). To test this hypothesis, we compared the protein expression status of ACE2 by immunohistochemistry (IHC) in post-mortem lung samples of patients who died of severe COVID-19 and lung samples obtained from non-COVID-19 patients for other indications. IHC for CD61 and CD163 was performed for the assessment of platelet-rich microthrombi and macrophages, respectively. IHC for SARS-CoV-2 viral antigen was also performed. In a total of 55, 44 COVID-19 post-mortem lung samples were tested for ACE2, 36 for CD163, and 26 for CD61, compared to 15 non-covid 19 control lung sections. Quantification of immunostaining, random sampling, and correlation analysis were used to substantiate the morphologic findings. Our results show that ACE2 protein expression was significantly higher in COVID-19 post-mortem lung tissues than in controls, regardless of sample size. Histomorphology in COVID-19 lungs showed diffuse alveolar damage (DAD), acute bronchopneumonia, and acute lung injury with SARS-CoV-2 viral protein detected in a subset of cases. ACE2 expression levels were positively correlated with increased expression levels of CD61 and CD163. In conclusion, our results show significantly higher ACE2 protein expression in severe COVID-19 disease, correlating with increased macrophage infiltration and microthrombi, suggesting a pathobiological role in disease severity.

Alterations of CD163 expression in the complications of diabetes: A systematic review.

AIMS: Diabetes mellitus is a state of chronic low-grade inflammation. Scavenger receptor CD163, expressed on monocyte/macrophage cells with anti-inflammatory functions, has been observed in diabetes complications. This review aimed to systematically survey human studies published until 31st January 2022 for CD163 expression, in particular diabetes complications and additionally to investigate whether CD163 may be implicated as a biomarker of, and mediator in, the progression of diabetes complications. METHODS: A systematic literature search undertaken in Scopus, Embase and Medline established 79 papers of relevance. Data extraction and assessment followed the PRISMA workflow. RESULTS: Based on specific criteria, 11 studies totalling 821 participants were included in this review. CD163 was quantified in various forms including soluble, cell surface, and mRNA measures. This review found that soluble CD163 was upregulated in diabetes complications in various local body fluids and systemically in plasma or serum and therefore implicated in the progression of those complications. CD163+ cells and mRNA were variably expressed across diabetes complications. CONCLUSIONS: CD163 was altered in series of diabetes complications and the circulating sCD163 has potential utility as an inflammation biomarker. The variable expression of CD163 on cell surfaces and its mRNA across different diabetes complications warrants further systematic investigation.

Dynamic increase of M2 macrophages is associated with disease progression of colorectal cancers following cetuximab-based treatment.

We aimed to investigate the dynamic changes of gene expression profiles and immune microenvironment linked to resistance to cetuximab-based treatments in patients with metastatic colorectal cancer (mCRC). A total of 106 patients with RAS-wild type mCRC who were treated with cetuximab-based treatments were included as the study population. RNA-sequencing and multiplexed immunohistochemistry were performed using paired or unpaired pre-treatment and post-treatment tumor tissues. Differentially expressed gene analysis of paired pre-treatment and post-treatment tumor tissues that develop acquired resistance (AR) identified the AR signature. Gene ontology analysis of the AR signature indicated enrichment of immune-related pathway genes. Among the immune subsets whose abundance was estimated by CIBERSORT, M2 macrophages showed the most prominent positive correlation with the expression of the AR signature. Among the post-treatment samples, progressive disease (PD) tumors showed a significantly higher abundance of M2 macrophages compared to non-PD tumors. These findings were validated by multiplexed immunohistochemistry analysis: the density of CD68+CD206+ M2 macrophages significantly increased at the time of PD following cetuximab-based treatment, whereas it did not consistently change in the tumor pairs of non-PD. In conclusion, a dynamic increase of M2 macrophages is associated with disease progression during cetuximab-based treatment of mCRCs. Targeting M2 macrophages is a promising immunotherapeutic strategy in this clinical context.

Endoplasmic reticulum stress and the unfolded protein response in skeletal muscle of subjects suffering from peritoneal sepsis.

We provide a descriptive characterization of the unfolded protein response (UPR) in skeletal muscle of human patients with peritoneal sepsis and a sepsis model of C57BL/6J mice. Patients undergoing open surgery were included in a cross-sectional study and blood and skeletal muscle samples were taken. Key markers of the UPR and cluster of differentiation 68 (CD68) as surrogate of inflammatory injury were evaluated by real-time PCR and histochemical staining. CD68 mRNA increased with sepsis in skeletal muscle of patients and animals (p < 0.05). Mainly the inositol-requiring enzyme 1α branch of the UPR was upregulated as shown by elevated X-box binding-protein 1 (XBP1u) and its spliced isoform (XBP1s) mRNA (p < 0.05, respectively). Increased expression of Gadd34 indicated activation of PRKR-Like Endoplasmic Reticulum Kinase (PERK) branch of the UPR, and was only observed in mice (p < 0.001) but not human study subjects. Selected cell death signals were upregulated in human and murine muscle, demonstrated by increased bcl-2 associated X protein mRNA and TUNEL staining (p < 0.05). In conclusion we provide a first characterization of the UPR in skeletal muscle in human sepsis.

Inflammatory Gene Expression in Neck Perivascular and Subcutaneous Adipose Tissue in Men With Carotid Stenosis.

The inflammatory phenotype of neck adipose tissue (NAT) might reflect its involvement in the pathogenesis of carotid atherosclerosis. We investigated inflammatory gene expression in the subcutaneous and the perivascular (pericarotid) adipose tissue from patients with carotid stenosis (CS) undergoing endarterectomy and a control group of patients without significant carotid atherosclerosis undergoing thyroid surgery. Only male patients were included (n = 13 in each study group). Clinical and biochemical data along with serum leptin, adiponectin, and monocyte chemoattractant protein 1 (MCP-1) were collected. Adipose tissue samples were obtained from both the subcutaneous and pericarotid compartments. Real-time polymerase chain reaction was used to measure gene expression of macrophage markers and adipokines. The CS group had higher subcutaneous and pericarotid visfatin gene expression and higher pericarotid expression of MCP-1 and CD68 genes. The ratio between pericarotid CD206 and CD68 gene expression was similar between study groups. Adiponectin gene expression in both NAT compartments did not differ between groups, but it was negatively associated with body weight. These observations suggest that NAT, and especially the pericarotid compartment, express enhanced inflammatory properties in patients with CS, but the proportion of anti-inflammatory macrophages in advanced atherosclerosis seems to be maintained.