Itored during 72 h. Dose of infection and p.i. times were selected based on preliminary results and previous work [23].Plasma cytokine and chemokine levelsBlood was collected from 6 h-euthanized mice and stabilized with EDTA. Time p.i. was chosen based on previous studies [12,24]. Plasma supernatants were collected and kept at ?0uC. Plasmatic concentrations of TNF, IL-6, IFN-c, CCL2, CCL3, CCL4, CXCL1, and CXCL2 were determined using a custommade 8-plex cytokine Milliplex panel (Millipore) according to manufacturer’s instructions. Acquisition was performed on a Luminex 100 platform (Luminex) and analysis performed using Beadview Multiplex Data Analysis Software v.1.0 (Upstate/ Millipore).Statistical analysis Measurement of blood bacterial loadsFollowing infection, blood bacteremia was assessed in surviving mice by collecting a 5 ml blood sample from the tail up to day 6 p.i. Proper dilutions were plated and counted as described above.Data are presented as the mean 6 standard error of the mean or geometric mean with 95 confidence interval where appropriate. Unpaired t-tests where appropriate were performed to find statistical differences between groups. The Kaplan eier methodTLR2-Independent Activation by S. suisTable 1. Primer sequences used for Fexinidazole real-time RT-qPCR.Gene Cxcl1 (Kc) Cxcl2 (Mip2a) Il6 Ifng Ccl2 (Mcp1) Ccl3 (Mip1a) Ccl4 (Mip1b) Tnf Actinb B2mGenebank ID NM_008176 NM_009140 NM_031168 NM_008337 NM_011333 NM_011337 NM_013652 NM_013693 NM_007393 NM_Amplicon Size 101 bp 102 bp 139 bp 90 bp 108 bp 91 bp 109 bp 103 bp 170 bp 110 bpForward Sequence TCTCCGTTACTTGGGGACAC AACATCCAGAGCTTGAGTGTGA ATGGATGCTACCAAACTGGAT TGAGCTCATTGAATGCTTGG ATTGGGATCATCTTGCTGGT GTGGAATCTTCCGGCTGTAG GAAACAGCAGGAAGTGGGAG AGGGTCTGGGCCATAGAACT CCAACCGTGAAAAGATGACC ATGGCTCGCTCGGTGACCCTReverse Sequence CCACACTCAAGAATGGTCGC TTCAGGGTCAAGGCAAACTT TGAAGGACTCTGGCTTTGTCT ACAGCAAGGCGAAAAAGGAT CCTGCTGTTCACAGTTGCC ACCATGACACTCTGCAACCA CATGAAGCTCTGCGTGTCTG CCACCACGCTCTTCTGTCTAC AGCATAGCCCTCGTAGATG TTCTCCGGTGGGTGGCGTGASpan Intron Yes Yes Yes Yes Yes Yes Yes Yes Yes YesPCR Efficiency 103 100 103 97 103 95 95 99 98 99doi:10.1371/journal.pone.0065031.tand log-rank Peptide M chemical information Mantel-Cox tests were used to compare the survival rates of the studied groups. P,0.05 was considered statistically significant.Microarray data accession numberAll microarray raw data are available and have been deposited in the Gene Omnibus Expression database under accession number GSE45861 and GSE45862.Results TLR2-dependent or ndependent survival during S. suis infectionAs shown in Figure 1A, WT mice infected with the S. suis ST1 strain started dying at 20 h p.i. and 45 had succumbed by 72 h. On the other hand, TLR22/2 mice infected with the same strain showed no mortality at the same time point (P = 0.004), even up to 96 h p.i. (data not shown). Survival of WT and TLR22/2 mice after infection with the ST7 strain showed no significant differences (Fig. 1B). Indeed, WT and TLR22/2 mice started dying at 16 h p.i. and 60 of mice had succumbed by 72 h (P = 0.745). These data confirmed the higher virulence of the ST7 strain compared to the ST1 strain, especially during the first hours of infection, as previously observed in C57BL/6 mice [24]. Results obtained suggest that mouse mortality after infection with the ST1 strain is, at least partially, related to the presence of TLR2. On the other hand, high mortality generated by the ST7 strain was shown to be independent of this receptor. Similar results were.Itored during 72 h. Dose of infection and p.i. times were selected based on preliminary results and previous work [23].Plasma cytokine and chemokine levelsBlood was collected from 6 h-euthanized mice and stabilized with EDTA. Time p.i. was chosen based on previous studies [12,24]. Plasma supernatants were collected and kept at ?0uC. Plasmatic concentrations of TNF, IL-6, IFN-c, CCL2, CCL3, CCL4, CXCL1, and CXCL2 were determined using a custommade 8-plex cytokine Milliplex panel (Millipore) according to manufacturer’s instructions. Acquisition was performed on a Luminex 100 platform (Luminex) and analysis performed using Beadview Multiplex Data Analysis Software v.1.0 (Upstate/ Millipore).Statistical analysis Measurement of blood bacterial loadsFollowing infection, blood bacteremia was assessed in surviving mice by collecting a 5 ml blood sample from the tail up to day 6 p.i. Proper dilutions were plated and counted as described above.Data are presented as the mean 6 standard error of the mean or geometric mean with 95 confidence interval where appropriate. Unpaired t-tests where appropriate were performed to find statistical differences between groups. The Kaplan eier methodTLR2-Independent Activation by S. suisTable 1. Primer sequences used for real-time RT-qPCR.Gene Cxcl1 (Kc) Cxcl2 (Mip2a) Il6 Ifng Ccl2 (Mcp1) Ccl3 (Mip1a) Ccl4 (Mip1b) Tnf Actinb B2mGenebank ID NM_008176 NM_009140 NM_031168 NM_008337 NM_011333 NM_011337 NM_013652 NM_013693 NM_007393 NM_Amplicon Size 101 bp 102 bp 139 bp 90 bp 108 bp 91 bp 109 bp 103 bp 170 bp 110 bpForward Sequence TCTCCGTTACTTGGGGACAC AACATCCAGAGCTTGAGTGTGA ATGGATGCTACCAAACTGGAT TGAGCTCATTGAATGCTTGG ATTGGGATCATCTTGCTGGT GTGGAATCTTCCGGCTGTAG GAAACAGCAGGAAGTGGGAG AGGGTCTGGGCCATAGAACT CCAACCGTGAAAAGATGACC ATGGCTCGCTCGGTGACCCTReverse Sequence CCACACTCAAGAATGGTCGC TTCAGGGTCAAGGCAAACTT TGAAGGACTCTGGCTTTGTCT ACAGCAAGGCGAAAAAGGAT CCTGCTGTTCACAGTTGCC ACCATGACACTCTGCAACCA CATGAAGCTCTGCGTGTCTG CCACCACGCTCTTCTGTCTAC AGCATAGCCCTCGTAGATG TTCTCCGGTGGGTGGCGTGASpan Intron Yes Yes Yes Yes Yes Yes Yes Yes Yes YesPCR Efficiency 103 100 103 97 103 95 95 99 98 99doi:10.1371/journal.pone.0065031.tand log-rank Mantel-Cox tests were used to compare the survival rates of the studied groups. P,0.05 was considered statistically significant.Microarray data accession numberAll microarray raw data are available and have been deposited in the Gene Omnibus Expression database under accession number GSE45861 and GSE45862.Results TLR2-dependent or ndependent survival during S. suis infectionAs shown in Figure 1A, WT mice infected with the S. suis ST1 strain started dying at 20 h p.i. and 45 had succumbed by 72 h. On the other hand, TLR22/2 mice infected with the same strain showed no mortality at the same time point (P = 0.004), even up to 96 h p.i. (data not shown). Survival of WT and TLR22/2 mice after infection with the ST7 strain showed no significant differences (Fig. 1B). Indeed, WT and TLR22/2 mice started dying at 16 h p.i. and 60 of mice had succumbed by 72 h (P = 0.745). These data confirmed the higher virulence of the ST7 strain compared to the ST1 strain, especially during the first hours of infection, as previously observed in C57BL/6 mice [24]. Results obtained suggest that mouse mortality after infection with the ST1 strain is, at least partially, related to the presence of TLR2. On the other hand, high mortality generated by the ST7 strain was shown to be independent of this receptor. Similar results were.
