Myeloid-Specific SIRT6 Deletion Protects Against Particulate Matter (PM_2.5)-Induced Airway Inflammation

Figures & data

Table 1 Sequence of Primers Were Used in This Study

Gene	Primer Sequence (5’-3’)
SIRT6 (h)	F: CCCACGGAGTCTGGACCAT R: CTCTGCCAGTTTGTCCCTG
IL-6 (h)	F: CCTGAACCTTCCAAAGATGGC R: TTCACCAGGCAAGTCTCCTCA
TNF-α (h)	F: AGCTGGAGAAGGGTGACCGA R: CACAGGGCAATGATCCCAAAG
CXCL1 (h)	F: ACAGTGTGTGGTCAACATTTCTCAT R: GCCAGAAACACTGTAAAACTACCAT
CXCL2 (h)	F: CACAGTGTGTGGTCAACATTTCTC R: CTCTCTGCTCTAACACAGAGGGA
GAPDH (h)	F: TGTTGCCATCAATGACCCCTT R: CTCCACGACGTACTCAGCG
Sirt6(m)	F: ATGTCGGTGAATTATGCAGCA R: GCTGGAGGACTGCCACATTA
IL-6 (m)	F: TCTATACCACTTCACAAGTCGGA R: GAATTGCCATTGCACAACTCTTT
TNF-α(m)	F: CTGAACTTCGGGGTGATCGG R: GGCTTGTCACTCGAATTTTGAGA
Cxcl1(m)	F: CTGGGATTCACCTCAAGAACATC R: CAGGGTCAAGGCAAGCCTC
Cxcl2(m)	F: TGTCCCTCAACGGAAGAACC R: CTCAGACAGCGAGGCACATC
β-actin(m)	F: AGAGGGAAATCGTGCGTGAC R: CAATAGTGATGACCTGGCCGT
LysMCre-WT(m)	TTACAGTCGGCCAGGCTGAC
LysMCre-Mutant(m)	CCCAGAAATGCCAGATTACG
LysMCre-Common(m)	CTTGGGCTGCCAGAATTTCTC
Sirt6 gene deletion(m)	F: AGTGAGGGGCTAATGGGAAC R: AACCCACCTCTCTCCCCTAA

Abbreviations: h, Human; m, Mouse.

Figure 1 PM_2.5 exposure induced SIRT6 expression in macrophages. The expression of SIRT6 in lung tissues of mice was analyzed by RT-PCR (A) and Western blot (B). THP1 Cells were exposed to PM_2.5 at indicated times or concentrations, SIRT6 expression was analyzed by RT-PCR and Western blot (C–E). BMDMs were exposed to PM_2.5 at indicated times or concentrations, SIRT6 expression was analyzed by RT-PCR and Western blot (F–H). Data are presented as the mean ± SEM of three independent experiments. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

Abbreviations: PM, Particulate matter; SIRT6, Sirtuin 6; BMDMs, bone marrow–derived macrophages.

Figure 2 PM_2.5 induced inflammatory cytokines production in macrophages. THP1 cells and BMDMs were treated with PM_2.5 for 4 hours at indicated concentrations (25, 50, 75, 100μg/mL). The mRNA expression of Il-6, Tnf-α, Cxcl1, and Cxcl2 were increased in THP1 cells (A–D) and BMDMs (E–H) was analyzed by RT-PCR. Data are presented as the mean ± SEM of three independent experiments. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

Abbreviations: PM, Particulate matter; BMDMs, bone marrow–derived macrophages; Il-6, interleukin 6; Tnf-α, tumor necrosis-alpha; Cxcl, C-X-C ligand.

Figure 3 Knockdown of SIRT6 reduced PM_2.5-induced inflammatory cytokines in THP1 cells. THP1 cells were transfected with control siRNA and Sirt6 siRNA for 24 h, and then were treated with PM_2.5 at 100 μg/mL for 4 h. SIRT6 expression was measured using Western blot (A) and RT-PCR (B). The mRNA expression of Il6, Tnf-α, Cxcl1, and Cxcl2 in THP1 cells were determined using RT-PCR (C–F). Data are presented as the mean ± SEM of three independent experiments. **P<0.01, ***P<0.001, and ****P<0.0001.

Abbreviations: PM, Particulate matter; Il-6, interleukin 6; Tnf-α, tumor necrosis-alpha; Cxcl, C-X-C ligand.

Figure 4 Sirt1-deficient BMDMs attenuated PM_2.5-induced inflammatory cytokines. Genotyping was assessed in BMDMs from Sirt6^fl/fl and Sirt6^fl/fl-LysMCre mice using Western blot (A) and RT-PCR analysis (B). BMDMs were treated with PM_2.5 at 100 μg/mL for 4 h to measure the levels of Il6, Tnf-α, Cxcl1, and Cxcl2 using RT-PCR (C–F). Data are presented as the mean ± SEM of three independent experiments. ***P<0.001 and ****P<0.0001.

Abbreviations: PM, Particulate matter; Il-6, interleukin 6; Tnf-α, tumor necrosis-alpha; Cxcl, C-X-C ligand.

Figure 5 Conditional knockout of SIRT6 in macrophages attenuated PM_2.5-induced inflammatory responses in mice. The expression of SIRT6 in the lung tissue was assessed by Western blot (A). The number of total cells (B), macrophage (C), neutrophil (D), lymphocytes (E) and eosinophil (F) in the BALF were measured by Flow cytometry. Representative images of macrophages and neutrophils in the BALF were analyzed using Giemsa’s staining (G, Scale bar: 100 μm). Representative images of lung sections with hematoxylin and eosin (H&E) staining (H, Scale bar: 100 μm). Semi quantification of inflammation expression in the lungs were performed using Image Pro 6.1 software (I). Il6, Tnf-α, Cxcl1 and Cxcl2 mRNA expression in lung homogenate were measured by QPCR (J–M). Protein concentrations of cytokines above in lung homogenate were measured by ELISA (N–Q). Data are presented as Mean ± SEM of three independent experiments (n=6 for each group). *P<0.05 and **P<0.01.

Abbreviations: BALF, Bronchoalveolar lavage fluid; Il-6, interleukin 6; Tnf-α, tumor necrosis-alpha; Cxcl, C-X-C ligand.