The Role of Pulmonary and Systemic Immunosenescence in Acute Lung Injury
Brandenberger Christina1,2,3,*, Kling Katharina Maria1,2, Vital Marius4, Christian Mühlfeld1,2,3
1Institute of Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany 2Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DZL), Hannover, Germany 3Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy), Hannover, Germany 4Microbial Interactions and Processes Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany
Acute lung injury (ALI) is associated with increased morbidity and mortality in the elderly (> 65 years), but the knowledge about origin and effects of immunosenescence in ALI is limited. Here, we investigated the immune response at pulmonary, systemic and cellular level in young (2-3 months) and old (18-19 months) C57BL/6J mice to localize and characterize effects of immunosenescence in ALI. ALI was induced by intranasal lipopolysaccharide (LPS) application and the animals were sacrificed 24 or 72 h later. Pulmonary inflammation was investigated by analyzing histopathology, bronchoalveolar lavage fluid (BALF) cytometry and cytokine expression. Systemic serum cytokine expression, spleen lymphocyte populations and the gut microbiome were analyzed, as well as activation of alveolar and bone marrow derived macrophages (BMDM) in vitro. Pulmonary pathology of ALI was more severe in old compared with young mice. Old mice showed significantly more inflammatory cells and pro-inflammatory cyto- or chemokines (TNFα, IL-6, MCP-1, CXCL1, MIP-1α) in the BALF, but a delayed expression of cytokines associated with activation of adaptive immunity and microbial elimination (IL-12 and IFNγ). Alveolar macrophages, but not BMDM, of old mice showed greater activation after in vivo and in vitro stimulation with LPS. No systemic enhanced pro-inflammatory cytokine response was detected in old animals after LPS exposure, but a delayed expression of IL-12 and IFNγ. Furthermore, old mice had less CD8+ T-cells and NK cells and more regulatory T-cells in the spleen compared with young mice and a distinct gut microbiome structure. The results of our study show an increased alveolar macrophage activation and pro-inflammatory signaling in the lungs, but not systemically, suggesting a key role of senescent alveolar macrophages in ALI. A decrease in stimulators of adaptive immunity with advancing age might further promote the susceptibility to a worse prognosis in ALI in elderly.
Representative micrographs of pulmonary histological sections stained with H&E. The lung parenchyma of young (A) and old control animals (B) do not show any inflammatory cells. Cell infiltrates, such as neutrophils and macrophages were found 24 h and 72 h after LPS-treatment in young (C, E) and old LPS (D, F) mice, respectively. Arrows indicate inflammatory cell infiltrates in the parenchyma and arrow heads in the peribronchiolar and -vascular area. Inflammatory cells were more pronounced in old compared with young LPS-mice and increased over time. Scale bar = 100 µm. a = airway, v = blood vessel
Figure 2 Protein concentration and differential cells counts in the BALF
Protein concentration (A), total cells (B), neutrophils (C), macrophages (D), lymphocytes (E) and eosinophils (F) were assessed in the BALF. BALF protein concentration, total BALF cells and BALF neutrophil numbers were greater after 24 h of LPS exposure in old mice (data published in Kling et al. 2017 ) and after 72 h of exposure time the numbers of BALF macrophages were also induced in old compared with young mice. Numbers of BALF lymphocytes increased from 24 h to 72 h in young and old mice. Detection of BALF eosinophils was marginal. Each data point represents one animal; means are expressed by horizontal bars; lines indicate statistically significant (p < 0.05) differences between groups.
A) BALF Cytokines
Young 24 h
Old 24 h
Young 72 h
Old 72 h
165 / 20
340 / 25
63 / 28
230 / 50
437 / 56
1591 / 282
1044 / 381
6190 / 1428
20 / 3
201 / 53
53 / 14
269 / 43
686 / 116
1038 / 112
709 / 212
1300 / 242
82 / 8
353 / 32
53 / 14
120 / 23
18 / 5
0 / 0
25 / 13
84 / 27
719 / 205
4 / 1
346 / 95
326 / 80
7 / 1
10 / 2
9 / 2
14 / 3
7 / 2
2 / 0
14 / 9
78 / 32
Young 24 h
Old 24 h
Young 72 h
Old 72 h
10.0 / 0.8
12.1 / 3.0
7.2 / 1.8
8.1 / 2.0
45.7 / 7.2
40.9 / 7.9
21.0 / 8.4
54.9 / 17.1
1.8 / 0.5
0.2 / 0.1
1.7 / 0.5
1.5 / 0.7
16.8 / 5.6
0.1 / 0.0
5.3 / 1.9
1.5 / 0.4
Table 1 BALF and serum cytokines.
Figure 3 Activation of BALF AM after 24 h and 72 h exposure time
CD80 expression (A and B) and CD86 expression (C and D) were assessed in BALF AM from young and old control and LPS mice after 24 h and 72 h of exposure time. AM of old mice showed enhanced CD80 and CD86 expression compared with young mice after LPS exposure at both time points. Note: 24 h and 72 h experiments are not directly comparable since two protocols with different staining compensations were applied. Data points from 24 h experiments each represent the result of two pooled BALF samples (see material and methods). Means are expressed by horizontal bars; lines indicate statistically significant (p < 0.05) differences between groups.
Figure 4 Lymphocyte populations in the spleen
Lymphocyte populations were assessed in spleens of control and LPS-mice 72 h after LPS exposure. Percentage of CD25+ T-regs was elevated in the spleens of old mice (A), whereas CD8+ T-cells and NK cells were reduced (B and C). No significant changes were detected with LPS exposure in the percentages of T-regs and CD8+ T-cells. However, a decline with LPS exposure was measured in percentage of total NK cells in old mice and mature NK cells in both age groups (D). Each data point represents one animal; means are expressed by horizontal bars; lines indicate statistically significant (p < 0.05) differences between groups.
Figure 5 Composition of gut microbiota
Caecal microbiota composition was analyzed in young and old control mice. Nonmetric multidimensional scaling (NMDS) of bacterial communities for young mice (empty triangle) and old mice (filled triangle) are shown (A). Age significantly influenced community structure based on permutational ANOVA analysis (p<0.01). Ellipses represent the 95% confidence interval on standard errors of points. The composition of the caecal bacterial community at genus level (B) is given with specific taxa that significantly differed with age (a) based on FDR corrected bootstrapped Mann-Whitney U tests). uc: unclassified.
Figure 6 In vitro stimulation of AM and BMDM
AM and BMDM were stimulated for 24 h with 100 ng/mL LPS. CD80 and CD86 expression of in vitro stimulated AM was greater in cells from old compared with young donors (A and B). No difference was measured in TNFα secretion in the supernatant, but enhanced levels of MIP-1α in AM from old mice (C and D). In comparison, CD80 and CD86 expression was lower in LPS exposed BMDM from old compared with young mice (E and F). No difference was measured in cytokine expression in old compared with young BMDM (G and H). Each data point indicates an individual measurement; means are expressed by horizontal bars; lines indicate statistically significant (p<0.05) differences between groups.
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