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. 2022 Jan 27;13(1):86. doi: 10.1038/s41419-022-04535-z

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — A Flow cytometric quantification of the percentage of gated CD69⁺NK1.1⁺ among freshly isolated CD45⁺CD3-NK1.1⁺ splenocytes in n = 3 Young, n = 3 Old-GFP, and n = 3 Old-LAV-BPIFB4 mice. B Flow cytometric quantification of the CD206⁺/CD86⁺ ratio (indicative of the macrophage polarization into a M2 or M1 macrophage-like phenotype) among total CD11b⁺F4/80⁺ splenocytes in n = 3 Young, n = 3 Old-GFP and n = 3 Old-LAV-BPIFB4 mice. Results from each treatment group are expressed as mean ± SD. Pairwise comparisons statistically significant are indicated (*P < 0.05, **P < 0.01). Statistical evaluation was carried out by unpaired t-test (GraphPad® Prism). C ELISA quantification of inflammatory cytokines levels in splenocytes conditioned media of n = 3 Old-AAV-LAV-BPIFB4 mice as against those of n = 3 Old-GFP mice (*P < 0.05, **P < 0.01). Results are expressed as mean ± SD. Pairwise comparisons statistically significant are indicated (*P < 0.05, **P < 0.01). Statistical evaluation was carried out by ordinary one-way ANOVA corrected for Tukey’s multiple comparisons test (GraphPad® Prism). D Histogram shows the amount of CD45⁺F4/80⁺ CD38⁺ spleen-resident macrophages in n = 3 aged mice infected with AAV-LAV-BPIFB4 compared to n = 3 mice with GFP infection (*P < 0.05). Results are expressed as mean ± SD in terms of relative change to maximize the effect compared to their absolute percentage values (Supplementary Fig. 4). Pairwise comparisons statistically significant are indicated (*P < 0.05). Statistical evaluation was carried out by ordinary one-way ANOVA corrected for Tukey’s multiple comparisons test (GraphPad® Prism). E Bar graph shows the amount of CD45⁺F4/80-Ly6C⁺CD38⁺ spleen-resident monocytes in n = 3 aged mice infected with AAV-LAV-BPIFB4 compared to n = 3 GFP-infected mice (**P < 0.01) expressed as mean ± SD in terms of relative change. Pairwise comparisons statistically significant are indicated (**P < 0.01). Statistical evaluation was carried out by ordinary one-way ANOVA corrected for Tukey’s multiple comparisons test (GraphPad® Prism). F Frequency of CD45⁺F4/80-Ly6C⁺CD38⁺ spleen-resident monocytes stratified into Ly6C^high and Ly6C^low from n = 3 Old-AAV-LAV-BPIFB4 mice vs n = 3 Old-GFP mice is reported (****P < 0.001). Results are expressed as mean ± SD. Pairwise comparisons statistically significant are indicated (****P < 0.0001). Statistical evaluation was carried out by ordinary one-way ANOVA corrected for Tukey’s multiple comparisons test (GraphPad® Prism).