NK cells promote neutrophil recruitment in the brain during sepsisinduced. neuroinflammation
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1 NK cells promote neutrophil recruitment in the brain during sepsisinduced neuroinflammation Hao He 1, Tingting Geng 1, Piyun Chen 1, Meixiang Wang 1, Jingxia Hu 1, Li Kang 1, Wengang Song 1, * & Hua Tang 1, * 1 Institute of Immunology, Taishan Medical University, Taian, Shandong, , China Supplementary Fig. 1. Expression of chemokines for attracting neutrophils in the brain. Twelve hours after LPS or PBS treatment, mrna (n=6 per group) was extracted from the whole brain of mice. qpcr was performed to detect the expression of chemokines for attracting neutrophils. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < , ANOVA. Means ± SD are shown. Data shown are representative of at least 2 independent experiments. 1
2 Supplementary Fig. 2. Expression of proinflammatory cytokines by NK cells cocultured with microglia in vitro. CD3 - CD19 - NK1.1 + NK cells ( ) sorted from bone marrow in naïve mice were cocultured with or without microglia ( ) sorted from mice treated with PBS or LPS for 3 days. Eleven hours later, NK cells in the coculture were sorted again by flow cytometry for mrna extraction and subsequent cytokine analysis by qpcr. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < , ANOVA. Means ± SD are shown. Data shown are representative of at least 2 independent experiments. Supplementary Fig. 3. Depletion of NK cells does not regulate the expression of rate-limiting enzymes for synthesis or degradation of acetylcholine, gammaaminobutyric acid and catecholamine. Mice with or without depletion of NK cells were treated with PBS or LPS for 3 and 4 days. mrna was extracted from the whole 2
3 brain (n=4 6 per group) and used for analysis of the expression of rate-limiting enzymes for neurotransmitter synthesis and degradation by qpcr. (a) Choline acetyltransferase (ChAT) is for acetylcholine synthesis; glutamate decarboxylase forward (GAD) is for GABA synthesis; tyrosine hydroxylase (TH) is for catecholamine synthesis; (b) Acetylcholinesterase (AChE) is for acetylcholine degradation; GABA-transaminase (GABA-T) is for GABA degradation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < , ANOVA. Means ± SD are shown. All data in this figure are representative of at least 2 independent experiments. Supplementary Fig. 4. Regulation of depression-like behavior by NK cells in lowgrade CLP. Mice with or without depletion of NK cells received surgery and were divided into sham group and low-grade CLP group. On day 2 and day 7, sucrose preference test was performed to evaluate the depression-like behavior of mice from different groups (n=8 per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < , ANOVA. Means ± SD are shown. Data shown are representative of at least 2 independent experiments. 3
4 Supplementary Fig. 5. Dynamic changes of NK cells and neutrophils in the blood and brain during LPS-induced inflammation. At indicated time point after LPS treatment, mice (n=4 per group) were killed for assessment of NK cell and neutrophils in the blood and brain. Histogram shows the percentage and cell number of CD19 - CD3 - NK1.1 + NK cells and CD11b + Ly6C - Gr1 + neutrophils gated in CD45 + leukocytes. Supplementary Fig. 6. Microglia could not attract NK cells in the early stage of neuroinflammation. Twenty-one hours after PBS or LPS treatment, microglia ( ) were sorted from brain in PBS-treated mice and LPS-treated mice. Then in vivo recruitment assay was performed to detect the attraction of NK cells by microglia (n=5 6, per group). ANOVA. Means ± SD are shown. Data shown are representative of at least 2 independent experiments. 4
5 Supplementary Table S1. Primers used for qpcr analysis Gene Forward Primer Reverse Primer Gapdh TGTGTCCGTCGTGGATCTGA TTGCTGTTGAAGTCGCAGGAG Cxcl1 TGCACCCAAACCGAAGTCAT CTCCGTTACTTGGGGACACC Cxcl2 CCAACCACCAGGCTACAGG GCGTCACACTCAAGCTCTG Cxcl3 GAAAGGAGGAAGCCCCTCAC ACACATCCAGACACCGTTGG Cxcl4 TGGTCCCGAAGAAAGCGATG TTCAGGGTGGCTATGAGCTG Cxcl5 CACTCGCAGTGGAAAGAACG CGTGGGTGGAGAGAATCAGC Cxcl7 ATTGCAACGGAAATCGCCTG TGTTGCAAAGGTTGCTTGGAA Cxcl9 GGAGTTCGAGGAACCCTAGTG GGGATTTGTAGTGGATCGTGC Cxcl10 CCAAGTGCTGCCGTCATTTTC GGCTCGCAGGGATGATTTCAA Cxcl11 GGCTTCCTTATGTTCAAACAGGG GCCGTTACTCGGGTAAATTACA Cxcl12 AGAAACCTTCCACCAGAGCAG GCCGGATCTTGTGTTGAGTGA Ccl3 TTCTCTGTACCATGACACTCTGC CGTGGAATCTTCCGGCTGTAG Ccl4 TTCCTGCTGTTTCTCTTACACCT CTGTCTGCCTCTTTTGGTCAG Ccl5 GCTGCTTTGCCTACCTCTCC TCGAGTGACAAACACGACTGC Ccl8 TCTACGCAGTGCTTCTTTGCC AAGGGGGATCTTCAGCTTTAGTA Cx3cl1 ACGAAATGCGAAATCATGTGC CTGTGTCGTCTCCAGGACAA Xcl1 TTTGTCACCAAACGAGGACTAAA CCAGTCAGGGTTATCGCTGTG Il-1β GCAACTGTTCCTGAACTCAACT ATCTTTTGGGGTCCGTCAACT Il-6 TCCAGTTGCCTTCTTGGGAC GTGTAATTAAGCCTCCGACTTG Tnf-α ATGTCGGCTCCAGGACCTTA GGTAGTAACTGTTGACACCCACT Ifn-γ ACAGCAAGGCGAAAAAGGATG TGGTGGACCACTCGGATGA AChE ACCGATACTCTGGACGAGGC CCTGCTTGCTATAGTGGTCG ChAT CCATGACTGACCACAAGGCT TCAATGGCCATGCCGGTTAT TPH2 CTGAATCCGCCTGAGAGCAT CCGTACATGAGGACTCGGTG TH TACTTTGTGCGCTTCGAGGT GGAACCTTGTCCTCTCTGGC GAD CTGTCCCTGTGTGACAACCA TGGTAAGCTGCTTTGGCTCG MAO-A GCTTATGTGGGACCAACCCA GGAAATGCACCACGGAATGG MAO-B TCCACATTGACCAGACAGGG CTTCATGCCCAAAGCAGGTG SERT GCGACGTGAAGGAAATGCTG GGAGTTGGGGTGGACTCATC GABA-T GAACACTGGGGCTTGGATGA TTGGCCGAAACTCCTCCTTG AChE, Acetylcholinesterase; ChAT, choline acetyltransferase; TPH2, Neuronal Tryptophan hydroxylase 2; TH, tyrosine hydroxylase; GAD, Glutamate decarboxylase; MAO-A, Monoamine oxidase A; MAO-B, Monoamine oxidase B; 5
6 SERT, serotonin transporter; GABA-T, GABA-transaminase 6
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