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. 2023 Jun 29:14:1202009.
doi: 10.3389/fimmu.2023.1202009. eCollection 2023.

Airway macrophages display decreased expression of receptors mediating and regulating scavenging in early cystic fibrosis lung disease

Affiliations

Airway macrophages display decreased expression of receptors mediating and regulating scavenging in early cystic fibrosis lung disease

Lisa J M Slimmen et al. Front Immunol. .

Abstract

Background: Cystic fibrosis (CF) airway disease is characterized by chronic inflammation, featuring neutrophil influx to the lumen. Airway macrophages (AMs) can promote both inflammation and resolution, and are thus critical to maintaining and restoring homeostasis. CF AM functions, specifically scavenging activity and resolution of inflammation, have been shown to be impaired, yet underlying processes remain unknown. We hypothesized that impaired CF AM function results from an altered expression of receptors that mediate or regulate scavenging, and set out to investigate changes in expression of these markers during the early stages of CF lung disease.

Methods: Bronchoalveolar lavage fluid (BALF) was collected from 50 children with CF aged 1, 3 or 5 years. BALF cells were analyzed using flow cytometry. Expression levels of surface markers on AMs were expressed as median fluorescence intensities (MFI) or percentage of AMs positive for these markers. The effect of age and neutrophilic inflammation, among other variables, on marker expression was assessed with a multivariate linear regression model.

Results: AM expression of scavenger receptor CD163 decreased with age (p = 0.016) and was negatively correlated with BALF %neutrophils (r = -0.34, p = 0.016). AM expression of immune checkpoint molecule SIRPα also decreased with age (p = 0.0006), but did not correlate with BALF %neutrophils. Percentage of AMs expressing lipid scavenger CD36 was low overall (mean 20.1% ± 16.5) and did not correlate with other factors. Conversely, expression of immune checkpoint PD-1 was observed on the majority of AMs (mean PD-1pos 72.9% ± 11.8), but it, too, was not affected by age or BALF %neutrophils. Compared to matched blood monocytes, AMs had a higher expression of CD16, CD91, and PD-1, and a lower expression of CD163, SIRPα and CD36.

Conclusion: In BALF of preschool children with CF, higher age and/or increased neutrophilic inflammation coincided with decreased expression of scavenger receptors on AMs. Expression of scavenging receptors and regulators showed a distinctly different pattern in AMs compared to blood monocytes. These findings suggest AM capacity to counter inflammation and promote homeostasis reduces during initiation of CF airway disease and highlight new avenues of investigation into impaired CF AM function.

Keywords: airway macrophages; children; cystic fibrosis; inflammation; lung disease; neutrophils; resolution; scavenger receptor.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Lung disease parameters in CF patients ages 1, 3 and 5 years in I-BALL cohort (A) Structural lung damage across age, measured using the Perth Rotterdam Annotated Grid Morphometric Analysis for CF (PRAGMA-CF) score (n = 31). PRAGMA-CF %Disease (PRAGMA %Dis) is calculated as %bronchiectasis + %mucus plugging + %airway wall thickening and expressed as percentage of total lung volume. (B) Frequency of neutrophils in BALF (BALF %Neu) was calculated from flow cytometric data as the percentage CD66b+ neutrophils of total BALF CD45+ leukocytes (gating strategy provided in Supplementary Figure 1 ). Dots represent individual subjects, and the horizontal line in each graph the median. ns, p > 0.05, * p < 0.05, **p < 0.005, ***p < 0.0001.
Figure 2
Figure 2
CD163 expression on airway macrophages decreases with age and increased neutrophilic inflammation. Airway macrophages (AMs) were identified as CD45+ CD66b- CD115+ CD33+ cells. Frequency of neutrophils in BALF (BALF %Neu) was calculated from flow cytometric data as the percentage CD66b+ neutrophils of total BALF CD45+ leukocytes (gating strategy provided in Supplementary Figure 1 ). (A) Representative flow cytometry plot depicting CD163 expression on AMs in a 1 year old subject and a 5 year old subject. CD163 expression levels were expressed as either the median fluorescent intensity (MFI) of the total AM population (B, D), or the percentage of AMs positive for CD163 (C, E). (B, C) Expression of CD163 on AMs across age groups. Kruskal-Wallis test and Mann-Whitney test were used to compare age groups. Dots represent individual subjects, and the horizontal line in each graph the median (n = 45 for MFI, n = 50 for percentages). (D-E) Spearman correlation was used to assess the correlations between BALF %Neu and CD163 expression. Lines depict linear regression. ns, p > 0.05, *p < 0.05, **p < 0.005.
Figure 3
Figure 3
CD91 expression on airway macrophages trends towards decrease with neutrophilic inflammation but not with age. Airway macrophages (AMs) were identified as CD45+ CD66b- CD115+ CD33+ cells. Frequency of neutrophils in BALF (BALF %Neu) was calculated from flow cytometric data as the percentage CD66b+ neutrophils of total BALF CD45+ leukocytes (gating strategy provided in Supplementary Figure 1 ). CD91 expression levels were expressed as either the median fluorescent intensity (MFI) of the total AM population (A, C), or the percentage of AMs positive for CD91 (B, D). (A, B) Expression of CD91 on AMs across age groups. Dots represent individual subjects, and the horizontal line in each graph the median (n = 44 for MFI, n = 48 for percentages). Kruskal-Wallis test and Mann-Whitney test were used to compare age groups. (C, D) Spearman correlation was used to assess the correlations between BALF %Neu and CD91 expression. Lines depict linear regression. ns, p > 0.05.
Figure 4
Figure 4
SIRPα expression on airway macrophages decreases with age and but not with neutrophilic inflammation. Airway macrophages (AMs) were identified as CD45+ CD66b- CD115+ CD33+ cells. Frequency of neutrophils in BALF (BALF %Neu) was calculated from flow cytometric data as the percentage CD66b+ neutrophils of total BALF CD45+ leukocytes (gating strategy provided in Supplementary Figure 1 ). (A) Representative flow cytometry plot depicting SIRPα expression on AMs in a 1 year old subject and a 5 year old subject. SIRPα expression levels were expressed as either the median fluorescent intensity (MFI) of the total AM population (B, D), or the percentage of AMs positive for SIRPα (C, E). (B, C) Expression of SIRPα on AMs across age groups. Dots represent individual subjects, and the horizontal line in each graph the median (n = 44 for MFI, n = 48 for percentages). Kruskal-Wallis test and Mann-Whitney test were used to compare age groups. (D, E) Spearman correlation was used to assess the correlations between BALF %Neu and SIRPα expression. Lines depict linear regression. ns, p > 0.05, **p < 0.005, ***p < 0.0001.
Figure 5
Figure 5
Expression of other phagocytosis-related and immune checkpoint markers on AMs. Frequency of neutrophils in BALF (BALF %Neu) was calculated from flow cytometric data as the percentage CD66b+ neutrophils of total BALF CD45+ leukocytes (gating strategy provided in Supplementary Figure 1 ). Airway macrophages (AMs) were identified as CD45+ CD66b- CD115+ CD33+ cells. Marker expression levels were expressed as either the median fluorescent intensity (MFI) of the total AM population (C), or the percentage of AMs positive for SIRPα (A, B, D). Dots represent individual subjects, and the horizontal line in each graph the median (n = 44-45 for MFI, n = 49-50 for percentages). (A) Expression of CD36 on AMs across age groups. (B, C) Expression of CD16 across age groups and BALF %Neu. (D) Expression of PD-1 on AMs across age groups. Kruskal-Wallis test and Mann-Whitney test were used to compare age groups. Spearman correlation was used to assess the correlations between BALF %Neu and CD16 expression. Lines depict linear regression. ns, p > 0.05, *p < 0.05.
Figure 6
Figure 6
Expression of SRs and phagocytosis-related markers on AMs differs from that on blood monocytes. Airway macrophages (AM) in BALF were identified as CD45+ CD66b- CD115+ CD33+ cells. Blood monocytes (BM) were identified as CD45+ FSCINT SSCINT CD66b- CD115+ cells (gating strategies provided in Supplementary Figures 1, 2 ). Surface marker expression was expressed as the median fluorescent intensity (MFI) of the total AM or blood monocyte population. Dots represent individual subjects, and the horizontal line in each graph the median. (A-G) comparisons of respective marker expressions on AM and BM (n = 44-45 for BALF, n = 39 for blood, for CD47 analysis n = 20 for BALF and n = 10 for blood). Wilcoxon’s test was used for comparison between AM and BM. ns, p > 0.05, *p < 0.005, ***p < 0.0001.

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