inhibition of toll-like receptor signaling by an itim … of toll-like receptor signaling by an...
TRANSCRIPT
Inhibition of toll-like receptor signaling by an ITIM-containing bacterial protein
Dapeng Yan1,5, Xingyu Wang2,3, Lijun Luo1, Xuetao Cao4 & Baoxue Ge1,2,3
1Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences/Shanghai Jiaotong University School of Medicine, Shanghai, China 2Clinical and Translational Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China 3Department of Microbiology and Immunology, Tongji University School of Medicine, Shanghai, China 4National Key Laboratory of Medical Immunology and Institute of Immunology, Second Military Medical University, Shanghai, China 5Graduate School of Chinese Academy of Sciences, Beijing, China
Correspondence should be addressed to B. Ge. ([email protected])
Supporting Online Materials
Supplementary Figures 1-10
Nature Immunology doi:10.1038/ni.2417
Supplementary Figure 1 Interaction of EHEC Tir and EBV LMP2A with SHP-1 (a,
b) Immunoprecipitation (IP) and immunoblot (IB) analysis of cell lysates from
HEK293T cells transfected with the indicated vectors using indicated antibodies. Data
are representative of at least three independent experiments.
Supplementary Figure 2 ITIM dependent interaction of EHEC Tir with SHP-1 (a)
Schematics of EHEC Tir ITIM mutants. (b) IP and IB analysis of cell lysates from
HEK293T cells transfected with the SHP-1 and various EHEC Tir ITIM mutants.
Data are representative of at least three independent experiments.
Supplementary Figure 3 Tir don’t inhibit activation of MAPK or NF-κB in the early
infection of EPEC. Immunoblot of cell lysates from peritoneal macrophages infected
with JPN15 or JPN15 ∆Tir for indicated times. Data are representative of three
independent experiments.
Supplementary Figure 4 Status of Tir phosphorylation in EPEC bacteria or in
EPEC-infected cells (a) JPN15 ∆Tir or JPN15 (∆Tir+HA-Tir) bacteria were broken by
ultra sonication, and the cell lysate were immunoprecipitate by HA antibody and
analyzed by antibody to p-Tyr or HA. IP and IB analysis of Raw264.7 cells infected
with the JPN15 ∆Tir or the JPN15 (∆Tir+HA-Tir) strains for the indicated time. (b) IP
and IB analysis of Raw264.7 cells infected with the JPN15 ∆Tir or the JPN15
(∆Tir+HA-Tir) strains for the indicated times. Data are representative of three
independent experiments.
Supplementary Figure 5 Phosphorylation of Tir in infected Hela cells. Confocal
microscope analysisi of HeLa cells infected with the indicated bacterial strains for
indicated times. Representative pictures were shown.
Supplementary Figure 6 T3SS-dependent inhibitory function of Tir (a) Immunoblot
of cell lysates from Raw264.7 cells infected with the E2348/69 or E2348/69 ∆ escN
Nature Immunology doi:10.1038/ni.2417
strains for indicated times. (b) Immunoblot of cell lysates from Raw264.7 cells
infected with the E2348/69 ∆ escN strains expressing Tir or its ITIM mutant for
indicated times. (c, d) Quantitative RT-PCR of Tnf or Il6 mRNA in Raw264.7 cells
infected with different E2348/69 strains for the indicated times. **P < 0.01
(Student's t-test). Data are representative of at least three independent experiments
(mean and s.e.m. in c and d).
Supplementary Figure 7 Tir specifically inhibits the cytokine protein production (a,
b) ELISA of TNF (a) and IL-6 (b) in supernatants of primary macrophages infected
medium or infected with JPN15, JPN15 ∆Tir, or JPN15 (∆Tir+HA-Tir) strains for 6
hours. **P < 0.01 (Student's t-test). Data are representative of at least three
independent experiments (mean and s.e.m.).
Supplementary Figure 8 ITIM-dependent inhibition of cyotkoine protein production
by Tir (a, b) ELISA of TNF (a) and IL-6 (b) in supernatants of primary macrophages
infected medium or infected with JPN15 ∆Tir strains expressing HA-Tir or Tir ITIM
mutants for 6 hours. **P < 0.01 (Student's t-test). Data are representative of at least
three independent experiments (mean and s.e.m.).
Supplementary Figure 9 Tir inhibits peritoneal immunity against C. rodentium. (a)
Survival rate of mice infected i.p. with Citrobacter rodentium or Citrobacter
rodentium ∆Tir strains. (1.25х109 CFU; n = 10 per group). (b) The bacteria count in
peritoneal lavage fluid from mice survived in a. *P < 0.05 (Student’s t-test). (c) The
bacteria count in peritoneal lavage fluid from mice infected with Citrobacter
rodentium or Citrobacter rodentium ∆Tir strains after administration of 108
CFU/mouse. *P < 0.05 and **P < 0.01 (Student's t-test). Data are representative of at
least three independent experiments (mean and s.e.m. in b and c).
Supplementary Figure 10 Diagram depicting the negative regulation of toll-like
signaling pathways by EPEC Tir.
Nature Immunology doi:10.1038/ni.2417
Nature Immunology doi:10.1038/ni.2417
Nature Immunology doi:10.1038/ni.2417
Nature Immunology doi:10.1038/ni.2417
Nature Immunology doi:10.1038/ni.2417
Nature Immunology doi:10.1038/ni.2417
Nature Immunology doi:10.1038/ni.2417
Nature Immunology doi:10.1038/ni.2417
Nature Immunology doi:10.1038/ni.2417
Nature Immunology doi:10.1038/ni.2417
Nature Immunology doi:10.1038/ni.2417