To Örebro University

Ageing is associated with dysregulated immune responses, resulting in impaired resilience against infections and low-grade inflammation known as inflammageing. Frailty is a measurable condition in older adults characterized by decreased health and physical impairment. Dendritic cells (DCs) and monocytes play a crucial role in initiating and steering immune responses. To assess whether their frequencies and phenotypes in the blood are affected by ageing or frailty, we performed a flow cytometry study on monocyte and DC subsets in an immune ageing cohort. We included (n = 15 in each group) healthy young controls (HYC, median age 29 years), healthy older controls (HOC, 73 years) and Frail older controls (76 years). Monocyte subsets (classical, intermediate, non-classical) were identified by CD14 and CD16 expression, and DC subsets (conventional (c)DC1, cDC2, plasmacytoid (p)DC) by CD11c, CD1c, CD141 and CD303 expression. All subsets were checked for TLR2, TLR4, HLA-DR, CD86, PDL1, CCR7 and CD40 expression. We observed a lower proportion of pDCs in HOC compared to HYC. Additionally, we found higher expression of activation markers on classical and intermediate monocytes and on cDC2 in HOC compared to HYC. Frail participants had a higher expression of CD40 on classical and non-classical monocytes compared to the HOC group. We document a substantial effect of ageing on monocytes and DCs. Reduced pDCs in older people may underlie their impaired ability to counter viral infections, whereas enhanced expression of activation markers could indicate a state of inflammageing. Future studies could elucidate the functional consequences of CD40 upregulation with frailty.

In atherosclerotic lesions, monocyte-derived macrophages are major source of interferon gamma (IFN-γ), a pleotropic cytokine known to regulate the expression of numerous genes, including the antiviral gene RSAD2. While RSAD2 was reported to be expressed in endothelial cells of human carotid lesions, its significance for the development of atherosclerosis remains utterly unknown. Here, we harnessed publicly available human carotid atherosclerotic data to explore RSAD2 in lesions and employed siRNA-mediated gene-knockdown to investigate its function in IFN-γ-stimulated human aortic smooth muscle cells (hAoSMCs). Silencing RSAD2 in IFN-γ-stimulated hAoSMCs resulted in reduced expression and secretion of key CXCR3-chemokines, CXCL9, CXCL10, and CXCL11. Conditioned medium from RSAD2-deficient hAoSMCs exhibited diminished monocyte attraction in vitro compared to conditioned medium from control cells. Furthermore, RSAD2 transcript was elevated in carotid lesions where it was expressed by several different cell types, including endothelial cells, macrophages and smooth muscle cells. Interestingly, RSAD2 displayed significant correlations with CXCL10 (r =  0.45, p = 0.010) and CXCL11 (r = 0.53, p = 0.002) in human carotid lesions. Combining our findings, we uncover a novel role for RSAD2 in hAoSMCs, which could potentially contribute to monocyte recruitment in the context of atherosclerosis.

Trimethylamine-N-oxide (TMAO) is a gut microbiota-derived metabolite and TNF-α is proinflammatory cytokine, both known to be associated with renal inflammation, fibrosis and chronic kidney disease. However, today there are no data showing the combined effect of TMAO and TNF-α on renal fibrosis-and inflammation. The aim of this study was to investigate whether TMAO can enhance the inflammatory and fibrotic effects of TNF-α on renal fibroblasts. We found that the combination of TNF-α and TMAO synergistically increased fibronectin release and total collagen production from renal fibroblasts. The combination of TMAO and TNF-α also promoted increased cell proliferation. Both renal proliferation and collagen production were mediated through Akt/mTOR/ERK signaling. We also found that TMAO enhanced TNF-α mediated renal inflammation by inducing the release of several cytokines (IL-6, LAP TGF-beta-1), chemokines (CXCL-6, MCP-3), inflammatory-and growth mediators (VEGFA, CD40, HGF) from renal fibroblasts. In conclusion, we showed that TMAO can enhance TNF-α mediated renal fibrosis and release of inflammatory mediators from renal fibroblasts in vitro. Our results can promote further research evaluating the combined effect of TMAO and inflammatory mediators on the development of kidney disease.

Laminins are essential components of the basement membranes, expressed in a tissue- and cell-specific manner under physiological conditions. During inflammatory circumstances, such as atherosclerosis, alterations in laminin composition within vessels have been observed. Our study aimed to assess the influence of tumor necrosis factor-alpha (TNF), a proinflammatory cytokine abundantly found in atherosclerotic lesions, on endothelial laminin gene expression and the effects of laminin-332 (LN332) on endothelial cells' behavior. We also evaluated the expression of LN332-encoding genes in human carotid atherosclerotic plaques. Our findings demonstrate that TNF induces upregulation of LAMB3 and LAMC2, which, along with LAMA3, encode the LN332 isoform. Endothelial cells cultured on recombinant LN332 exhibit decreased claudin-5 expression and display a loosely connected phenotype, with an elevated expression of chemokines and leukocyte adhesion molecules, enhancing their attractiveness and adhesion to leukocytes in vitro. Furthermore, LAMB3 and LAMC2 are upregulated in human carotid plaques and show a positive correlation with TNF expression. In summary, TNF stimulates the expression of LN332-encoding genes in human endothelial cells and LN332 promotes an endothelial phenotype characterized by compromised junctional integrity and increased leukocyte interaction. These findings highlight the importance of basement membrane proteins for endothelial integrity and the potential role of LN332 in atherosclerosis.

BACKGROUND: The formation and accumulation of cholesterol crystals (CC) at the lesion site is a hallmark of atherosclerosis. Although studies have shown the importance of vascular smooth muscle cells (VSMCs) in the disease atherosclerosis, little is known about the molecular mechanism behind the uptake of CC in VSMCs and their role in modulating immune response.

METHODS: Human aortic smooth muscle cells were cultured and treated with CC. CC uptake and CC mediated signaling pathway and protein induction were studied using flow cytometry, confocal microscopy, western blot and Olink proteomics. Conditioned medium from CC treated VSMCs was used to study neutrophil adhesion, ROS production and phagocytosis. Neutrophil extracellular traps (NETs) formations were visualized using confocal microscopy.

RESULTS: VSMCs and macrophages were found around CC clefts in human carotid plaques. CC uptake in VSMCs are largely through micropinocytosis and phagocytosis via PI3K-AkT dependent pathway. The uptake of CC in VSMCs induce the release inflammatory proteins, including IL-33, an alarming cytokine. Conditioned medium from CC treated VSMCs can induce neutrophil adhesion, neutrophil reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) formation. IL-33 neutralization in conditioned medium from CC treated VSMCs inhibited neutrophil ROS production and NETs formation.

CONCLUSION: We demonstrate that VSMCs due to its vicinity to CC clefts in human atherosclerotic lesion can modulate local immune response and we further reveal that the interaction between CC and VSMCs impart an inflammatory milieu in the atherosclerotic microenvironment by promoting IL-33 dependent neutrophil influx and NETs formation.

Background and Aims: Peripheral arterial disease (PAD) is a clinical manifestation of atherosclerosis, affecting arteries in the leg. Based on their symptoms and severity, PAD patients are characterized into three sub-groups: asymptomatic, intermittent claudication (IC) and critical limb ischemia (CLI). Despite its high prevalence, PAD remains under diagnosed and the role of immune cells in PAD pathophysiology remains poorly understood. In this study, we characterized the innate immune responses in PAD patients compared to healthy controls.

Methods: Blood samples were collected from 14 patients with PAD (IC) and 30 healthy controls, to assess the phenotype of monocytes and neutrophils by using 10-colour flow cytometry. Phagocytosis assay was performed with labelled E.coli particles. Mann-Whitney U non-parametrical test was used for statistical comparison between PAD patients and healthy controls.

Results: A significant higher proportion of leukocytes (p<0.05) and neutrophils (p<0.01) was observed in PAD patients compared to healthy controls, whereas monocyte subsets showed no significant differences. Interestingly, neutrophils showed a significantly impaired phagocytosis capability (p<0.05) and reduced expression of myeloperoxidase (MPO) (p<0.05) in PAD patients compared to healthy controls.

Conclusions: Taken together these results, suggest that PAD patients have an increased proportion of neutrophils in circulation, with impaired phagocytosis capability, compared to healthy controls.

BACKGROUND AND AIMS: Laminins are essential components of the endothelial basement membrane, which predominantly contains LN421 and LN521 isoforms. Regulation of laminin expression under pathophysiological conditions is largely unknown. In this study, we aimed to investigate the role of IL-6 in regulating endothelial laminin profile and characterize the impact of altered laminin composition on the phenotype, inflammatory response, and function of endothelial cells (ECs).

METHODS: HUVECs and HAECs were used for in vitro experiments. Trans-well migration experiments were performed using leukocytes isolated from peripheral blood of healthy donors. The BiKE cohort was used to assess expression of laminins in atherosclerotic plaques and healthy vessels. Gene and protein expression was analyzed using Microarray/qPCR and proximity extension assay, ELISA, immunostaining or immunoblotting techniques, respectively.

RESULTS: Stimulation of ECs with IL-6+sIL-6R, but not IL-6 alone, reduces expression of laminin α4 (LAMA4) and increases laminin α5 (LAMA5) expression at the mRNA and protein levels. In addition, IL-6+sIL-6R stimulation of ECs differentially regulates the release of several proteins including CXCL8 and CXCL10, which collectively were predicted to inhibit granulocyte transmigration. Experimentally, we demonstrated that granulocyte migration is inhibited across ECs pre-treated with IL-6+sIL-6R. In addition, granulocyte migration across ECs cultured on LN521 was significantly lower compared to LN421. In human atherosclerotic plaques, expression of endothelial LAMA4 and LAMA5 is significantly lower compared to control vessels. Moreover, LAMA5-to-LAMA4 expression ratio was negatively correlated with granulocytic cell markers (CD177 and myeloperoxidase (MPO)) and positively correlated with T-lymphocyte marker CD3.

CONCLUSIONS: We showed that expression of endothelial laminin alpha chains is regulated by IL-6 trans-signaling and contributes to inhibition of trans-endothelial migration of granulocytic cells. Further, expression of laminin alpha chains is altered in human atherosclerotic plaques and is related to intra-plaque abundance of leukocyte subpopulations.

Background and Aims: Peripheral arterial disease (PAD) is a common manifestation of atherosclerosis, affecting the lower limbs. T cells are among the principal contributors to the development of atherosclerotic plaques. However, T cell immune responses in PAD pathophysiology are poorly understood and a detailed phenotypic and functional characterization of T cell immune responses in PAD is needed.

Methods: Blood samples were collected from PAD patients with claudicatio intermittens (n=14) and healthy controls (HCs, n=30). We assessed the phenotype of active, effector and memory T cell subsets by evaluating the expression of specific surface and intracellular markers analysed by 10-colour flow cytometry. Functional responses were evaluated by performing T cell receptor (TCR) stimulation of PBMCs in a 3D cell culture system to assess cytokine production by ELISA. Statistical analyses were performed using the Mann-Whitney U test.

Results: No differences were observed between PAD and HCs in terms of active, effector and memory T cell phenotypes and in the frequency of cells expressing CCR6 and CXCR3 (markers associated with T cells producing IL-17 and IFN-γ). However, lower frequencies of IFN-γ+ cells among CD8+ (P=0.04), and CD4+CD8+ cells (P=0.03) were observed in PAD compared to HCs. TNF-α production in PAD-derived PBMCs, via TCR stimulation was increased at both 48- (P=0.004) and 72-hour time points (P=0.003). No differences were observed in IL-1β, IFN-γ and IL-17 secretion.

Conclusions: Taken together these results suggest that increased TNF-α secretion by PBMCs in response to TCR activation might contribute to the pro-inflammatory environment in PAD pathogenesis.

Background and Aims: LDL-c is cleared from the circulation mainly by hepatic LDL-receptor mediated endocytosis. Defective LDL-c clearance and hence its elevation in circulation is one of the risk factors for myocardial infarction (MI). A soluble LDL-R (sLDL-R) exists in human plasma and exhibits strong correlation with circulating LDL-c and conditions that promote chronic inflammation. However, the mechanistic interplay between sLDL-R, inflammation and MI remains to be investigated.

Methods: In vitro studies using HepG2 cells treated with TNF-α, and a nested case-control study was conducted to investigate the relationship between plasma sLDL-R, TNF-α and risk of future MI.

Results: Stimulation of HepG2 cells with TNF-α induces release of sLDL-R with limited effect on surface expression of LDL-R. TNF-α induces gene expression of peptidases ADAM17 and MMP14 in HepG2 cells, and inhibition of ADAM17 and MMP-14 significantly reduces the TNF-α induced sLDL-R release. Although TNF-α treatment of HepG2 cells has limited effect on LDL-c endocytosis, HepG2 cells incubated with recombinant sLDL-R showed reduced LDL-c uptake in a dose-dependent manner. In a nested case-control study, baseline sLDL-R in plasma was positively correlated with plasma total cholesterol level. Further, a 2-fold increase in plasma sLDL-R was associated with 2.1x higher risk of future MI. Using mediation analyses, we determined that significant proportion of the association is mediated by elevation in plasma cholesterol level.

Conclusions: Our study suggests that sLDL-R is generated by TNF-α via membrane shedding. Further, an increase in sLDL-R could inhibit hepatic clearance of LDL-c increasing its half-life in the circulation and contributing to the pathogenesis of MI.