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Supplemental digital content 7 (supplementary methods):
F-Actin assembly
Neutrophils from bone marrow were incubated with CXCL2 (30ng/ml) at
37°C for two minutes. Cells were fixed, permeabilized, and stained with rhodamine-phalloidin (Molecular Probes, USA). Microscopic analysis of fluorescent images was performed using an Olympus BX40-F4 epi-fluorescence microscope (400X). The mean fluorescence intensity was determined using
Image J software (Imaging process and analysis in Java, National Institute of
Mental Health, USA). Twenty cells from five randomly chosen fields were analyzed.
Killing
Neutrophils from BM (10 6 cells) were incubated with cecum-bacteria
(2x10 6 microorganisms
/cell) for two hours at 37°C. Next, the cells were washed with PBS, lysed with Triton 0.2% and sonicated for two minutes. The supernatant was plated on Muller-Hinton agar dishes (Difco Laboratories, USA) and incubated at 37°C. Colony forming units (CFU) were scored after 18 hours of culture.
Reactive oxygen species production
The hydrogen peroxide production was estimated by incubation of whole blood with 60 µM H
2
DCF-DA (2,7 dichlorofluorescein diacetate, Biotium, Inc.
Hyward, CA). The samples were acquired using a BD Biosciences FACScanto flow cytometer. The mean fluorescent intensity of DCF was determined in

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Gr1 high population using the FlowJo 7.6.5 software (Flow Cytometry Analysis
Software, Tree Star Inc., USA).
Intravital microscopy
Animals were anesthetized i.p. with ketamine (100 mg/kg) and xylazine
(10 mg/kg). The mesenteric tissue was exposed for microscopic examination in situ . The animals were maintained on a thermostatically board controlled at
37 °C, with a transparent platform on which the tissue to be transilluminated was placed. Blood vessels selected for study were third-order venules, defined according to their branch-order location within the microvascular network. The interaction of leukocytes with the luminal surface of the venular endothelium was studied in a segment of the vessel. A leukocyte was considered to be adherent to the venular endothelium if it remained stationary for >30 seconds.
Adherent cells (“stickers”) were expressed as the number per 0,1 mm 2 of venule.

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Supplemental Digital Content 1. Toll-like receptor 9 deficiency does not alter local chemokine production after severe sepsis (S-CLP). Wild-type
(WT) and TLR9 -/ mice were given severe sepsis (S-CLP). Six hours after CLP,
CXCL1, CXCL2, TNFα and IL-1β levels were measured in the peritoneal exudates by enzyme-linked immunosorbent assay (n=5-10). *p<0.05.
Supplemental Digital Content 2. Toll-like receptor 9 activation inhibits actin polymerization induced by CXCL2. Bone marrow neutrophils were incubated two hours with CpG oligodeoxinucleotide type B (CpG-B ODN;
0.3µg/ml) or lipopolysaccharide (LPS; 30 µg/mL) in culture medium (RPMI). The cells were stimulated with CXCL2 (30ng/ml). The figure is representative of immunofluorescence staining for rhodamine-phalloidin (red marker) merged with 4, 6-diamidino-2-phenylindole (DAPI; nuclear blue marker). The graph represents the mean fluorescence intensity (MFI) of 20 cells. *p<0.05.
Supplemental Digital Content 3. CpG-B but not CpG-A ODN inhibits neutrophil chemotaxis. Bone marrow neutrophils were incubated for one hour with lipopolysaccharide (LPS; 30µg/ml), Control ODN (0.3µg/ml), CpG oligodeoxynucleotide type B (CpGB ODN; 0.3µg/ml) or CpG oligodeoxynucleotide type A (CpG-A
ODN; 0.3µg/ml) in culture medium (RPMI).
The black bars represent the cells submitted to chemotaxis toward CXCL2
(30ng/ml) and the white bar represents chemotaxis toward culture medium
(RPMI). *p<0.05.

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Supplemental Digital Content 4. Toll-like receptor 9 activation in neutrophils does not interfere with cell viability. Bone marrow neutrophils were incubated for two or 24 hours with CpG oligodeoxynucleotide type B
(CpGB ODN; 0.3µg/ml) in culture medium (RPMI). The cells were stained with annexin V and propidium iodide (PI). The plots represent the percentage of viable cells (PI Annexin V ) in an experiment performed in triplicate. The double negative cells were quantified by flow cytometry. *p<0.05.
Supplemental Digital Content 5. Toll-like receptor 9 activation reduces neutrophil adherence. A: Wild-type (WT) mice were treated with CpG oligodeoxinucleotide type B (CpG-B ODN; 0.9mg/kg), lipopolysaccharide (LPS;
15mg/kg) or control oligodeoxinucleotide (control ODN; 0.9 mg/kg) and injected with thioglycollate i.p. After three hours, intravital microscopy was performed to measure the number of leukocytes adherent on mesentery endothelium. (n=3-
4). B and C: Wild-type (WT) and TLR9 -/ mice were given severe (S-CLP) or non severe sepsis (NS-CLP) by the cecal ligation and puncture (CLP). The expression levels of CD11b and CD18 were measured by flow cytometry (n=5-
7) twelve hours after CLP. D: Bone marrow neutrophils were incubated for two hours with CpG oligodeoxynucleotide type B (CpGB ODN; 0.3 µg/ml) in culture medium (RPMI), and the expression of CD11b was measured by flow cytometry. MFI: median fluorescence intensity. *p<0.05.
Supplemental Digital Content 6. Toll-like receptor 9 activation by neutrophils is important for killing bacteria but not indispensable. A: Bone marrow neutrophils were incubated for one hour with CpG oligodeoxynucleotide

5 type B (CpGB ODN; 0.3µg/ml) in culture medium (RPMI). The killing assay was performed three hours after incubation with cecal bacteria
(2microorganisms/cell). B: The killing assay was performed in neutrophils from
WT and TLR9 -/ mice. The pointed lines represent the bacterial input. CFU: colony forming units. C: DCF (dichlorofluorescein) was used to determine the production of reactive oxygen species by neutrophils (Gr1 high population) from
WT and TLR9 -/ mice with severe sepsis. MFI: mean fluorescence intensity. The results are representative of two independent experiments performed in triplicate. *p<0.05.