Phenotypic and functional characteristics of blood monocytes in chronic kidney disease

Human blood monocytes, recently classified as classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14-CD16++) are reported to be dysregulated in chronic kidney disease (CKD) and may play a role in CKD-associated cardiovascular disease and increased mortality risk. However, the phenotypic and functional properties of the monocyte subsets have not been fully characterised across the full range of CKD stages. The overall aim of this thesis was to methodically characterise major circulating immune cell populations with particular focus towards monocytes and their subsets as well as to determine if NLRP3 inflammasome status and monocyte responsiveness are altered in a clinically stable, ambulatory, non-dialysis requiring cohort of adults with CKD. This was performed by blood sampling and clinical data collection from a cohort of adults with medically stable CKD as well as healthy adults and included development of new methods for profiling and analysing the inflammatory functions of human blood monocytes. The first experimental section of the project aimed to: (a) accurately quantify major circulating leukocyte populations and monocyte subsets repertoires in CKD (b) profile a newly proposed intermediate monocyte subdivision based on dichotomous surface expression of human leukocyte antigen [(HLA)-DR] in CKD (c) examine the relationship between cell populations and the demographic, clinical and laboratory indices of a CKD cohort and (d) determined if these cell populations have a relationship with blood levels of CKD biomarkers and inflammatory mediators. The results (described in Chapter 3) showed that total blood monocyte and neutrophil (but not lymphocyte) numbers were higher in adults with CKD stages 2-5 compared to healthy adults and correlated with measures of kidney function. Among the monocyte subsets, it was found that the intermediate subset had the strongest relationship with CKD stage. In addition, a novel subpopulation of intermediate monocytes with high surface levels of (HLA)-DR was found to correlate most closely with kidney function. Serum concentrations of several inflammatory mediators and biomarkers were increased in a CKD stage-dependent manner and these correlated with intermediate monocyte and neutrophil number. The results of this section demonstrate relationships between circulating innate immune cells and CKD severity. Selective expansion of intermediate monocytes and increases in specific cytokines and biomarkers indicate that CKD-associated inflammation has distinct characteristics that may be of value for risk prediction and disease monitoring. The second experimental section of the project aimed to: (a) phenotypically compare scavenger receptor and TLR expression profiles of circulating monocytes in CKD (b) correlate these expression profiles with demographic, clinical and laboratory indices and (c) determine if expression of inflammsome-related gene products are altered in CKD. The results (described in Chapter 4) showed that monocyte surface expression of the pro-inflammatory ligand-gated cation channel, P2X7R and other proteins from the scavenger receptor family was higher in peripheral blood mononuclear cell (PBMC) samples from CKD subjects compared to those from healthy adults. However, in the same PBMC samples, monocyte expression of toll like receptors and genes related to the inflammasome complex were not increased in CKD. Taken together, these results indicate that, while altered monocyte expression of scavenger receptors may be characteristic of CKD, the inherent activation of monocyte inflammatory pathways that has been reported in end-stage renal disease patients receiving chronic dialysis therapy is not present in those with medically managed, non-dialysis requiring CKD. The third experimental section of the project aimed to: (a) characterise inflammasome activation in peripheral blood monocytes of healthy adults (b) determine whether monocyte inflammasome response is enhanced in CKD (c) define the relationships between monocyte inflammasome response and demographic, clinical and laboratory indices in CKD and (d) examine whether CKD-associated soluble serum factors enhance monocyte inflammasome response. The results (described in Chapter 5), revealed that freshly-isolated monocytes from healthy adults respond strongly to low concentrations of ligands for TLR4 and TLR7/8 and rapidly release interleukin 1β (IL-1β) in a P2X7R-dependent manner following addition of exogenous adenosine triphosphate (eATP). Release of IL-1β following TLR4 or TLR7/8 + eATP exposure, which is indicative of activation of the inflammasome complex, was greater for PBMC from adults with CKD stages 3 and 4 compared to those from healthy adults. Additionally, PBMC release of tumour necrosis factor α (TNFα) in response to TLR4 and TLR7/8 ligands was greater for CKD3/4 compared to healthy adults. Finally, stimulation experiments involving culture of PBMC with serum samples from CKD 3/4 or healthy subjects were performed. The results, although variable, suggested that CKD serum may contain factors which enhance inflammasome activation following exposure to TLR ligands and eATP. Taken together, the results for this section indicated that CKD is associated with altered monocyte inflammatory responsiveness involving multiple TLR pathways as well as the inflammasome. Overall, the results of this project add novel details to current knowledge regarding major circulating human leucocyte populations and monocyte subset repertoires in CKD and demonstrate links between these cellular alterations and the levels of inflammatory mediators and biomarkers in the circulation. They suggest that, in clinically stable adults with non dialysis-dependent CKD, circulating monocytes are not inherently activated but have alterations to scavenger receptor expression and heightened responsiveness to TLR- and inflammasome-activating stimuli. The results are of clinical relevance to the development of assays for more accurately profiling inflammatory status of those affected by CKD. They also have potential to better inform the design of clinical trials of novel therapies that target abnormal inflammation in CKD. Further studies to determine the mediators and mechanisms underlying enhanced monocyte responsiveness to inflammatory stimuli in CKD may result in the identification of new biomarkers or therapeutic targets for this common chronic disease.2021-02-1