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IntroductionCD5L is a scavenger receptor-like molecule that mediates diverse physiologic processes, including cell survival, atherogenesis, inflammation, and lipid metabolism. Even though CD5L is an abundant circulatory protein, it has recently become apparent that its expression can alter inflammatory signaling in a cell-autonomous fashion. To date, the effect of endogenous CD5L expression in human macrophages remains largely unexplored. Our work addressed this question by analyzing the impact of CD5L gene disruption on the inflammatory state of the THP-1 human monocytic cell line.ResultsIn macrophage-like CD5L-knockout cells, we observed a dramatic decrease in the basal expression of a subset of NF-κB-regulated genes when compared to control cell lines. These differences persisted after stimulation with lipopolysaccharide (LPS), even though the magnitude of induction was similar in both mutant and control cells. Consistent with the lipid remodeling function attributed to CD5L, we found significant changes in the makeup of the intracellular lipid pool. However, we did not detect significant changes in the activity of fatty acid synthase, which has been suggested to mediate CD5L lipidome remodeling function. Furthermore, we explored how CD5L function impacts undifferentiated monocytes. We found that in undifferentiated, unstimulated monocytes deleted for CD5L, several dysregulated transcripts code for genes involved in cell-to-cell interactions and in the progression of atherosclerosis. Most importantly, we found that CD5L deletion upregulates the expression of CD52, a novel anti-inflammatory switch.DiscussionOverall, our findings further support the multifunctional nature of CD5L and, for the first time, suggest its involvement in monocyte localization to sites of future lesions.

Cancer screening and diagnosis can be achieved by analyzing specific molecules within serum-derived extracellular vesicles (EVs). This study sought to profile EV-derived proteins to identify potential lung cancer biomarkers. EVs were isolated from 80 serum samples from healthy individuals and cancer patients via polyethylene glycol (PEG)-based precipitation and immunoaffinity separation using antibodies against CD9, CD63, CD81, and EpCAM. Proteomic analysis was performed using 2-D gel electrophoresis and matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI–TOF MS). The expression of proteins that were differentially upregulated in the EVs or tissue of lung cancer samples was validated by Western blotting. The area under the curve (AUC) was calculated to assess the predictability of each differentially expressed protein (DEP) for lung cancer. A total of 55 upregulated protein spots were selected, seven of which (CD5L, CLEC3B, ITIH4, SERFINF1, SAA4, SERFINC1, and C20ORF3) were found to be expressed at high levels in patient-derived EVs by Western blotting. Meanwhile, only the expression of EV CD5L correlated with that in cancer tissues. CD5L also demonstrated the highest AUC value (0.943) and was found to be the core regulator in a pathway related to cell dysfunction. Cumulatively, these results show that EV-derived CD5L may represent a potential biomarker—detected via a liquid biopsy—for the noninvasive diagnosis of lung cancer.

Introduction Resolution of acute gut ischemia causes reperfusion injury, resulting in the release of damage-associated molecular patterns (DAMPs) and tissue injury. A key DAMP, extracellular cold-inducible RNA-binding protein (eCIRP), exacerbates inflammation in reperfusion injury, contributing to organ failure and death. Apoptosis inhibitor of macrophage (AIM or CD5L) is a glycoprotein secreted by macrophages which can influence the activity of immune cells. We seek to investigate AIM expression in ischemia/reperfusion (I/R) and elucidate its anti-inflammatory role in macrophages and intestinal epithelial cells. Methods Male mice underwent occlusion of the superior mesenteric artery for 60 min, followed by reperfusion for 4 h before sample collection. AIM expression in blood and tissue was evaluated by qPCR, Western blot, and ELISA. Primary peritoneal macrophages from male mice, IEC-6 intestinal epithelial cells, and RAW 264.7 macrophages were stimulated with recombinant mouse (rm) CIRP (denoted eCIRP) and treated with rmAIM. Cytokine levels were assessed by ELISA and qPCR. Metabolic function was measured in macrophages using the Agilent Seahorse XF Pro analyzer. Interactions involving AIM, eCIRP, and eCIRP’s receptors, Toll-like receptor 4 (TLR4) and triggering receptor expressed on myeloid cells-1 (TREM-1), were elucidated by in silico approaches. Results Pulmonary AIM mRNA expression decreased by 55.9% ( p  = 0.018), and protein levels decreased by 26.9% ( p  = 0.032) in gut I/R mice compared to sham mice. Plasma AIM concentration decreased by 22.0% ( p  = 0.0362) in gut I/R mice compared to sham. eCIRP treatment increased pro-inflammatory cytokine production by macrophages and intestinal epithelial cells. This increase was significantly attenuated by co-treatment with rmAIM. Macrophages also increased basal oxygen consumption rate by 66.7% and ATP production by 70.3% when treated with rmAIM compared to eCIRP stimulation alone ( p  < 0.0001). Computational modeling predicted strong interactions between AIM and eCIRP’s receptors, TLR4 and TREM-1, and showed that the presence of AIM altered eCIRP’s binding to these receptors. Conclusion In male mice, gut I/R decreases AIM protein levels and mRNA expression in the lungs as well as AIM plasma concentration. AIM reduces eCIRP-induced pro-inflammatory cytokine production in macrophages, potentially by inhibiting eCIRP’s binding to TLR4 and TREM-1. These findings suggest AIM is a promising therapeutic candidate in males with gut I/R.

CD5L (CD5 molecule-like) is a secreted glycoprotein that controls key mechanisms in inflammatory responses, with involvement in processes such as infection, atherosclerosis, and cancer. In macrophages, CD5L promotes an anti-inflammatory cytokine profile in response to TLR activation. In the present study, we questioned whether CD5L is able to influence human macrophage plasticity, and drive its polarization toward any specific phenotype. We compared CD5L-induced phenotypic and functional changes to those caused by IFN/LPS, IL4, and IL10 in human monocytes. Phenotypic markers were quantified by RT-qPCR and flow cytometry, and a mathematical algorithm was built for their analysis. Moreover, we compared ROS production, phagocytic capacity, and inflammatory responses to LPS. CD5L drove cells toward a polarization similar to that induced by IL10. Furthermore, IL10- and CD5L-treated macrophages showed increased LC3-II content and colocalization with acidic compartments, thereby pointing to the enhancement of autophagy-dependent processes. Accordingly, siRNA targeting ATG7 in THP1 cells blocked CD5L-induced CD163 and Mer tyrosine kinase mRNA and efferocytosis. In these cells, gene expression profiling and validation indicated the upregulation of the transcription factor ID3 by CD5L through ATG7. In agreement, ID3 silencing reversed polarization by CD5L. Our data point to a significant contribution of CD5L-mediated autophagy to the induction of ID3 and provide the first evidence that CD5L drives macrophage polarization.

Background Apoptosis inhibitor of macrophage (AIM) is a fundamental mediator of recovery from acute kidney injury (AKI) in mice, but its role in AKI in dogs is unknown. Hypothesis/Objectives To detect and quantify AIM in serum and urine from both healthy dogs and dogs with AKI, and to determine if AIM is higher in dogs with AKI compared to healthy controls. Animals Eight dogs with International Renal Interest Society (IRIS) Grade II–V AKI and 10 healthy adult dogs. Methods Retrospective case–control study. Liquid chromatography‐mass spectrometry (LC–MS) based targeted proteomics was used to quantify AIM. AIM peak areas were compared between the AKI and healthy cohorts and correlated with selected markers of renal function. Results AIM was able to be quantified in the urine of 5/8 dogs with AKI and 1/10 healthy dogs. AIM was quantified in the serum of all dogs, and there was no difference in peak area between the two groups (AKI: median, 67 840 (range, 9797‐98 725); control: median, 79 072 (range, 46 400‐160 330); p = 0.274). In dogs with AKI, AIM was not correlated with serum creatinine, blood urea nitrogen, phosphorus, or potassium concentrations, urine specific gravity, or IRIS AKI grade. Conclusions and Clinical Importance AIM was predominantly detected in the urine of dogs with AKI and not in the urine of healthy dogs.

Sepsis, a life-threatening condition caused by a dysregulated host response to infection, remains a major cause of mortality worldwide. Identifying reliable biomarkers for prognosis and treatment is urgently needed. This study investigates the role of the Apoptosis Inhibitor of Macrophages (AIM), also known as CD5L, as a potential prognostic biomarker and therapeutic target in sepsis. We measured free and total AIM concentrations in 90 septic patients enrolled in SepsisDataNet.NRW cohort (German Clinical Trial Registry No. DRKS00018871; http://www.sepsisdatanet.nrw). Blood samples were collected on days 1, 4, and 8, and AIM levels were quantified using ELISA. Kaplan-Meier analysis and Cox regression were performed to assess the association between AIM levels and 30-day survival. Western blot analysis was performed to detect AIM in human serum IgM and in the IgM-enriched intravenous immunoglobulin IVIG preparation Pentaglobin . High total AIM concentrations (>85 ng/ml) were significantly associated with improved 30-day survival on day 1 (HR: 3.131, 95% CI: 1.629-6.019, p = 0.009), 4 (HR: 2.525, 95% CI: 1.198-5.322, p = 0.0042), and day 8 (HR: 2.317, 95% CI: 0.8565-6.266, p = 0.0457). Free AIM showed a significant association with survival only on day 8 (HR: 2.374, 95% CI: 0.8721-6.461, p = 0.0393). Total AIM concentration is a significant predictor of a 30-day survival in sepsis, supporting its potential use as a prognostic biomarker. Our findings also suggest that AIM may serve as a valuable prognostic biomarker and a potential target for immune-modulating therapies, including IgM-enriched intravenous immunoglobulins (IVIGs).

Plasma-derived vesicles hold a promising potential for use in biomedical applications. Two major challenges, however, hinder their implementation into translational tools: (a) the incomplete characterization of the protein composition of plasma-derived vesicles, in the size range of exosomes, as mass spectrometric analysis of plasma sub-components is recognizably troublesome and (b) the limited reach of vesicle-based studies in settings where the infrastructural demand of ultracentrifugation, the most widely used isolation/purification methodology, is not available. In this study, we have addressed both challenges by carrying-out mass spectrometry (MS) analyses of plasma-derived vesicles, in the size range of exosomes, from healthy donors obtained by 2 alternative methodologies: size-exclusion chromatography (SEC) on sepharose columns and Exo-Spin™. No exosome markers, as opposed to the most abundant plasma proteins, were detected by Exo-Spin™. In contrast, exosomal markers were present in the early fractions of SEC where the most abundant plasma proteins have been largely excluded. Noticeably, after a cross-comparative analysis of all published studies using MS to characterize plasma-derived exosomes from healthy individuals, we also observed a paucity of \"classical exosome markers.\" Independent of the isolation method, however, we consistently identified 2 proteins, CD5 antigen-like (CD5L) and galectin-3-binding protein (LGALS3BP), whose presence was validated by a bead-exosome FACS assay. Altogether, our results support the use of SEC as a stand-alone methodology to obtain preparations of extracellular vesicles, in the size range of exosomes, from plasma and suggest the use of CD5L and LGALS3BP as more suitable markers of plasma-derived vesicles in MS.

CD5L (CD5 molecular-like) plays an important role in lipid metabolism and immune regulation. This study aimed to investigate the roles of CD5L on liver hepatocellular carcinoma (LIHC). We analyzed the CD5L mRNA expression and its potential prognostic value based on The Cancer Genome Atlas and Gene Expression Omnibus databases. Immunohistochemical analysis was used to investigate the CD5L levels in LIHC tissues. Serum CD5L levels in LIHC were detected by enzyme-linked immunosorbent assay. Cell Counting Kit-8 (CCK-8) assay was used to investigate the effect of CD5L treatment on HepG2 and QSG-7701 cell proliferation. CD5L expression correlated genes were exhumed based on the LinkedOmics. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses for CD5L associated genes were performed. The correlation between CD5L and tumor immune infiltration was analyzed by using Tumor Immune Estimation Resource (TIMER) 2.0. CD5L mRNA and protein levels were significantly decreased in LIHC tumor tissue compared with non-tumor control tissues. Moreover, serum CD5L levels were significantly lower in LIHC patients than that in healthy subjects. Gene Expression Profiling Interactive Analysis 2 and Kaplan-Meier plotter analysis showed that a high-CD5L expression was correlated with favorable overall survival in LIHC patients, except the LIHC patients with hepatitis virus. CCK-8 results showed that CD5L treatment significantly decreased HepG2 cell proliferation in a concentration-dependent manner, and CD5L treatment had no effect on the proliferation of non-tumor hepatocyte line QSG-7701. CD5L associated genes were enriched in the immune response biological process, and CD5L expression levels were positively correlated with the immune infiltrates of CD8 + T cell and M1 macrophage cells but negatively correlated with CD4 + T cells and M0 macrophage cell infiltration. Exogenous CD5L inhibits cell proliferation of hepatocellular carcinoma. CD5L may act as a role of prognostic marker.

Fc receptor-like (FCRL) molecules comprise a large family of receptors, homologous to the receptors for the Fc portion of immunoglobulins (FCR). Within this family, an unusual gene known to exist in mice, rats and dogs, termed FCRLS , encodes a chimeric protein with both Ig-like FCRL and type B scavenger-receptor cysteine-rich (SRCR)-like domains. In mice, FCRLS is located next to the CD5L and KIRREL1 genes. Here, we show that the curious FCRLS gene is actually present across major mammalian groups, but its annotation is generally incorrect or absent. Anchored on mouse FCRLS and FCRL2 genomic sequence alignments, phylogenetic analyses demonstrated that many mammalian sequences currently annotated as FCRL2 cluster with FCRLS , supported by a conserved genetic synteny among organisms. This analysis shows that FCRLS is present in Rodentia, some Carnivora (Canidae and Ursidae), Chiroptera, Arctiodactyla, Proboscidae, and some Primata. Thus, the FCRLS most likely originated in a eutherian mammal ancestor since it is not present in Monotremata or Marsupialia. FCRLS has a peculiar distribution pattern across mammalian lineages, being present in some species, but absent in others from the same family, as in carnivores for example. The most parsimonious hypothesis to explain this FCRLS evolution is that it was convergently lost in several independent mammalian lineages. Analyses of branch-specific nucleotide evolutionary rates, show that FCRL2 and FCRLS have similar ranges of rates across mammals, suggesting that both genes have crucial, but separate functions in the immune system. Bayesian estimates of evolutionary rates for FCRLS in mammalian lineages revealed that carnivores display the highest mutation rate after rodents. Additionally, positive diversifying selection was detected for both FCRL2 and FCRLS . Our results show that the presence of the FCRLS gene is older and more widespread across mammals than previously thought and appears to be functional, being under positive selection. Its precise physiologic role should thus be investigated.

The defective eradication of invading pathogens is a major cause of death in sepsis. As professional phagocytic cells, macrophages actively engulf/kill microorganisms and play essential roles in innate immune response against pathogens. Growth differentiation factor 3 (GDF3) was previously implicated as an important modulator of inflammatory response upon acute sterile injury. In this study, administration of recombinant GDF3 protein (rGDF3) either before or after CLP surgery remarkably improved mouse survival, along with significant reductions in bacterial load, plasma pro-inflammatory cytokine levels, and organ damage. Notably, our in vitro experiments revealed that rGDF3 treatment substantially promoted macrophage phagocytosis and intracellular killing of bacteria in a dose-dependent manner. Mechanistically, RNA-seq analysis results showed that CD5L, known to be regulated by liver X receptor α (LXRα), was the most significantly upregulated gene in rGDF3-treated macrophages. Furthermore, we observed that rGDF3 could promote LXRα nuclear translocation and thereby, augmented phagocytosis activity in macrophages, which was similar as LXRα agonist GW3965 did. By contrast, pre-treating macrophages with LXRα antagonist GSK2033 abolished beneficial effects of rGDF3 in macrophages. In addition, rGDF3 treatment failed to enhance bacteria uptake and killing in LXRα-knockout (KO) macrophages. Taken together, these results uncover that GDF3 may represent a novel mediator for controlling bacterial infection.