Macrophage subpopulations in pediatric patients with lupus nephritis and other inflammatory diseases affecting the kidney

Abstract

Background: Macrophages play an important role in the pathogenesis of lupus nephritis (LN), but less is known about macrophage subtypes in pediatric LN. Here we compared renal inflammation in LN with other inflammatory pediatric kidney diseases and assessed whether inflammation correlates with clinical parameters.

Methods: Using immunofluorescence microscopy, we analyzed renal biopsies from 20 pediatric patients with lupus nephritis (ISN/RPS classes II-V) and pediatric controls with other inflammatory kidney diseases for infiltration with M1-like (CD68 + /CD206 - , CD68 + /CD163 -), M2a-like (CD206 + /CD68 +), and M2c-like macrophages (CD163 + /CD68 +) as well as CD3 + T-cells, CD20 + B-cells, and MPO + neutrophilic granulocytes. In addition, the correlation of macrophage infiltration with clinical parameters at the time of renal biopsy, e.g., eGFR and serum urea, was investigated. Macrophage subpopulations were compared with data from a former study of adult LN patients.

Results: The frequency of different macrophage subtypes in biopsies of pediatric LN was dependent on ISN/RPS class and showed the most pronounced M1-like macrophage infiltration in patients with LN class IV, whereas M2c-like macrophages were most abundant in class III and IV. Interestingly, on average, only half as many macrophages were found in renal biopsies of pediatric LN compared to adult patients with LN. The distribution of frequencies of macrophage subpopulations, however, was different for CD68 + CD206 + (M2a-like) but comparable for CD68 + CD163 - (M1-like) CD68 + CD163 + (M2c-like) cells in pediatric and adult patients. Compared to other inflammatory kidney diseases in children, fewer macrophages and other inflammatory cells were found in kidney biopsies of LN. Depending on the disease, the frequency of individual immune cell types varied, but we were unable to confirm disease-specific inflammatory signatures in our study due to the small number of pediatric cases. Worsened renal function, measured as elevated serum urea and decreased eGFR, correlated particularly strongly with the number of CD68 + /CD163 - M1-like macrophages and CD20 + B cells in pediatric inflammatory kidney disease.

Conclusion: Although M1-like macrophages play a greater role in pediatric LN patients than in adult LN patients, M2-like macrophages appear to be key players and are more abundant in other pediatric inflammatory kidney diseases compared to LN.

Keywords: Inflammatory kidney diseases; Lupus nephritis; Macrophage subtypes; Macrophages; Pediatric patients.

Fig. 1

Immunofluorescence staining of inflammatory cells in pediatric kidneys. A CD68 + macrophages. B CD163 + cells. C Merge of CD68 + and CD163 + cells representing M2c-like macrophages. D CD68 + macrophages. E CD 206 + cells. F Merge CD68 + and CD206 + cells, representing M2a-like macrophages. G Renal T lymphocytes (CD3 + cells). H Renal B lymphocytes (CD20 + cells). I B and T Lymphocyte overlap (CD3 + and CD20 + cells. Scale bar represents 50 µm

Fig. 2

Distribution of macrophage subsets in different ISN/RPS classes. A CD68 + macrophages in pediatric LN patient groups representing the total macrophages. B CD68 + CD206 − cells (M1-like macrophages) in pediatric LN patients. C CD68 + CD163 − cells (M1-like macrophages). D CD68 + as total macrophages in adult LN patients with a significant difference between groups II, V, and IV. E CD68 + CD206 − cells (M1-like macrophages) with a significant difference between groups IV and V. F CD 68 + CD163 − cells ((M1-like macrophages) (E and F in adult LN patients). G–I Showing results for pediatric LN patients, G CD68 + CD206 + cells (M2a-like macrophages), H CD68 + CD163 + cells (M2c-like macrophages), I Ration of CD68 + CD206 − vs CD68 + CD206 + cells (M1-like macrophages and M2a-like macrophages) with a significant difference between groups II and IV. J–L representing adult LN results. J CD68 + CD206 + cells (M2a-like macrophages), K CD68 + CD163 + cells (M2c-like macrophages), with a significant difference between groups II, V, and IV. L Ration of CD68 + CD206 − vs CD68 + CD206 + cells (M1-like macrophages and M2a-like macrophages). (*p < 0.05; **p < 0.01)

Fig. 3

Distribution of macrophage-subtypes in pediatric LN ISN/RPN class IV in glomeruli with and without (w/o) crescents. A Representative multiple immunofluorescence staining for CD68 + and CD163 + cells using biopsies from patients with ISN/RPN class IV. B Multiple immunofluorescence staining for CD68 + , CD206 + CD20 + , and CD3 + cells using the same glomerulus imaged in (A) in another section. C Pie chart showing the distribution of different inflammatory cells in glomeruli from pediatric LN biopsies with ISN/RPN class IV. D MPO + cells /glomerular cross-section (GCS). E CD3 + T-cells / GCS. F Glomerular CD20 + B-cells. G Glomerular total CD68 + macrophages per GCS (first antibody panel). H Glomerular CD68 + CD206 − M1-like macrophages/ GCS. I Glomerular CD68 + CD206 + M2a-like macrophages. J Ratio of glomerular CD68 + CD206 − /CD68 + CD206 + macrophages. K glomerular total CD68 + macrophages per GCS (second antibody panel). H Glomerular CD68 + CD163 − M1-like macrophages/ GCS. I Glomerular CD68 + CD163 + M2c-like macrophages. J Ratio of glomerular CD68 + CD163 − /CD68 + CD163 + macrophages. (*p < 0,05). Scale bar represents 50 µm

Fig. 4

Distribution of macrophage-subtypes in pediatric LN and a pediatric control group consisting of HUS, MPGN, PI-GN and PAUCI patients. A CD68 + total macrophages in the inflammatory kidney in pediatric patients with significantly higher in PAUCI patients compared to the LN and control group. B CD68 + CD206 − M1-like macrophages significantly higher in pediatric PAUCI patients compared to all other study groups. C CD68 + CD163 − M1-like macrophages significantly higher in PAUCI than in LN patients. D CD68 + CD206 + M2a-like macrophages, significantly higher in PAUCI than in LN patients. E CD68 + CD163 + M2c-like macrophages, significantly higher in HUS pediatric patients compared to MPGN, LN patients and our pediatric control group. F Ratio of CD68 + CD206 − and CD68 + CD206 + (M1-like and M2a-like macrophages) in pediatric patients. (*p < 0.05; **p < 0.01)

Fig. 5

T-, B-lymphocytes, and neutrophilic granulocytes in pediatric study groups. A CD3 + T-lymphocytes in pediatric patients. Significantly higher in PAUCI patients compared to the LN and control group. B CD20 + B-lymphocytes significantly higher in PAUCI and HUS compared to LN patients as well as post-infectious patients compared to our control group. C MPO + cells resembling neutrophils. (*p < 0.05;**p < 0.01)

Fig. 6

Clustering analysis of the abundance of inflammatory cells in different inflammatory kidney diseases in pediatric patients. Heatmap of the numbers of CD68 + CD163 + , CD68 + CD206 + , CD68 + CD163 − , CD68 + CD206 − , CD3 + , CD20 + , and MPO + cells in the analyzed patients (red: high number; blue: low number). The color-coded bar corresponding to the different rows (patients) indicates the diagnosis

Fig. 7

Glomerular injury score (GIS), serum creatinine, serum urea, and proteinuria used to evaluate the extent of kidney injury in pediatric patients with inflammatory kidney diseases. A GIS score 0. B GIS score 1. C GIS score 3. D GIS score 4. E GIS in pediatric patients. Significantly higher in all groups compared to the control group, significantly higher in PI-GN and PAUCI patients than in LN patients. F Serum creatinine is significantly higher in LN Patients compared to HUS and PAUCI patients. G Serum urea is significantly higher in pediatric HUS patients compared to LN patients and the control group. The control group as significantly lower levels than all study pathological groups. H Proteinuria in our study groups

Fig. 8

Correlation of clinical parameters with inflammatory cell infiltrates in pediatric inflammatory kidney disease. A Significant (p < 0.05) correlations between the eGFR, serum urea, and proteinuria as markers of kidney function with the cell counts of inflammatory cells including macrophage subtypes. B Significant (p < 0.05) correlations between cortical CD3 + T-cells, CD20 + , and MPO + cells with the cell counts of macrophage subtypes and subtype ratios detected in all pediatric inflammatory kidney disease groups, as assessed by Spearman’s test