Fig 1.
Anti-mouse TLR4/MD2 antibody clone MTS510 suppresses TLR4-mediated immune response in vitro.
Murine RAW264.7 macrophage cells were incubated with (or without) anti-TLR4/MD2 (clone MTS510) antibody for 30min before incubation of the cells for 2 days with 10ng/ml lipopolysaccharide (LPS). TNFα concentration in the supernatant was determined by the use of a bioassay with the L929 reporter cell line and supernatant (1:200 dilution). TNFα concentration is indicated in pg/ml and was determined with 4 measurements of three separate biological samples in each group (control: incubation without LPS and MTS510 antibody; LPS: incubation with LPS only; LPS+TLR4: incubation with MTS510 antibody before stimulation with LPS), data are present with the mean ± SD (***p< 0.0001 as determined by Mann-Whitney U test).
Fig 2.
Evaluation of the therapeutic potential of of anti-TLR4 mAb in vivo applied i.p..
MTS510 mAb (TLR4-Ab), or isotype control antibody (Iso) were applied i.p. at the end of 45min MCAO and once again after 24h of reperfusion in a dose of 1μg/animal in each application. A second control was performed by omission of any antibody and thus pure injection of vehicle (PBS). A correction for edema (brain swelling) was applied by calculating the indirect infarct volume as the volume of the contralateral hemisphere minus the non-infarcted volume of the ipsilateral hemisphere. The difference between direct (A) and indirect (B) infarct volumes represents brain swelling (C). Neurological deficit (D) was ranked according to a modified Bederson-score from 0 (no deficit) to 4 (dead). Data are presented as single values (scatter blots) combined with the mean ± SD; neuroscore graph shows single values and median (nIso = 10; nPBS = 10; nTLR4Ab = 8).
Fig 3.
Evaluation of the therapeutic potential of anti-TLR4 mAb in vivo applied i.a..
MTS510 mAb (TLR4-Ab), isotype control (Iso), or vehicle (PBS) was applied i.a. at the start of MCAO (45min) in a dose of 1μg/animal in each application. Besides application of isotype control antibody, a second control was performed by omission of any antibody and injection of vehicle only (PBS). The difference between direct (A) and indirect (B) infarct volumes at 48h of reperfusion represents brain swelling/edema (C). Neurological deficit (D) at 48h of reperfusion after 45min of MCAO was ranked according to a modified Bederson-score from 0 (no deficit) up to 4 (dead). Data are presented as single values (scatter blots) combined with the mean ± SD; neuroscore graph shows single values and the median (***p < 0.001; **p < 0.01; *p < 0.05; Mann-Whitney U test; number of animals in treatment groups: niso = 8, nPBS = 10, nTLR4Ab = 10).
Fig 4.
Evaluation of the therapeutic potential of anti-TLR4 mAb in vivo applied i.v..
MTS510 mAb (TLR4-Ab), respectively vehicle (PBS) was applied i.v. at the end of MCAO for 45min and once again after 24h of reperfusion in a dose of 1μg/animal each application. A second control was performed by omission of any antibody and pure injection of vehicle (PBS). The difference between direct (A) and indirect (B) infarct volumes represents brain swelling/edema (C). Neurological deficit (D) at 48 h of reperfusion was ranked according to a modified Bederson-score from 0 (no deficit) up to 4 (dead). Data are presented as single values (scatter blots) combined with the mean ± SD; neuroscore graph shows single values and the median (*p< 0.05; Mann-Whitney U) (nPBS = 12; nTLR4Ab = 15).
Fig 5.
Evaluation of inflammatory cell counts in whole hemispheres of i.v. vehicle, and anti-TLR4mAb treated wild-type mice at 48h after 45min MCAO.
Total cell count in each hemisphere was determined by the use of flow cytometry. CD11b-positive cells (activated macrophages/microglia) were shown with their absolute cell counts (A) in each, ispilateral/ischemic (ipsi) and contralateral/non-ischemic (contra) hemisphere of PBS- or anti-TLR4mAb-treated mice; as well as with the ratio of induction in ischemic hemisphere when compared to contralateral/non-ischemic hemisphere (B). Fig 5C shows the number of neutrophils identified in whole ischemic and non-ischemic hemispheres of PBS and anti-TLR4-mAb treated mice (mean values ± SD; *p< 0.05; Mann-Whitney U; nPBS = 6; nTLR4Ab = 5).
Fig 6.
Evaluation of cells of the adaptive immune response at 48h after 45min MCAO in mice i.v. treated with anti-TLR4mAb or vehicle control.
Through CD3- or CD45.B220-specific selection by the use of flow cytometry, the total T-cell count (A), and the total B-cell count (B) in whole ischemic (ipsi), and non-ischemic (contra) hemispheres of i.v. TLR4mAb treated (TLR4-Ab) and vehicle (PBS) treated mice was determined at 48h after 45min of MCAO (mean values ± SD; *p< 0.05; Mann-Whitney U; nPBS = 6; nTLR4Ab = 5).