Journal: Journal of Neuroinflammation

Article Title: Inflammatory monocytes and microglia play independent roles in inflammatory ictogenesis

doi: 10.1186/s12974-022-02394-1

Figure Lengend Snippet: Viral load in the brain is not reduced in the low inoculum group, but inflammatory chemokine and cytokine production are reduced by more than 90% at 1 dpi. A Viral load was assessed at 1, 3, and 7 dpi in mice inoculated with 200,000 PFU or 12,500 PFU of TMEV. Despite a 16-fold reduction in the amount of virus delivered to the brain, the groups exhibit similar viral loads at each timepoint. F (5,70) = 0.1534, P = 0.6964 between 200,000 PFU and 12,500 PFU by two-way ANOVA on ranks (inoculum x day); Shapiro–Wilk P = 0.0033; Tukey HSD pairwise analysis on ranks: 200,000 PFU vs 12,500 PFU @ 1 dpi: P = 0.6938; @ 3 dpi: P = 0.9900; @ 7 dpi: P = 0.9989; results from 5 separate experiments. B , C Representative images of TMEV immunostaining (green) in CA1 pyramidal neurons at 3 dpi in a mouse inoculated with 200,000 PFU ( B ) or 12,500 PFU ( C ) of virus; DAPI is shown in blue. Boxes indicate higher magnification insets “ i ” and “ ii ”. The amount of CCL2 ( D ), TNFα ( E ), and IL6 ( F ) in hippocampal homogenates was measured at 24 and 72 hpi. All of the factors were profoundly reduced at 24 hpi in the mice inoculated with 12,500 PFU of TMEV relative to mice inoculated with 200,000 PFU, with TNFα and IL6 indistinguishable from vehicle control mice. D CCL2, F (2,12) = 318.3, P < 0.0001 by two-way ANOVA; Shapiro–Wilk P = 0.8826 (24 hpi); Tukey’s pairwise analysis: 24 hpi: 200,000 PFU vs 12,500 PFU: P < 0.0001; 200,000 PFU vs vehicle: P < 0.0001; 12,500 PFU vs vehicle: P = 0.5123; 72 hpi: 200,000 PFU vs 12,500 PFU: P = 0.4512; 200,000 PFU vs vehicle: P = 0.9999; 12,500 PFU vs vehicle: P = 0.5252; d s @ 24 hpi = 11.8; results from 3 separate experiments. E TNFα, F (2,28) = 15.92, P < 0.0001 by two-way ANOVA; Shapiro–Wilk P = 0.7674; Tukey’s pairwise analysis: 24 hpi: 200,000 PFU vs 12,500 PFU: P < 0.0001; 200,000 PFU vs vehicle: P < 0.0001; 12,500 PFU vs vehicle: P > 0.9999; 72 hpi: 200,000 PFU vs 12,500 PFU: P = 0.1562; 200,000 PFU vs vehicle: P = 0.3342; 12,500 PFU vs vehicle: P > 0.9999; d s @ 24 hpi = 4.6; results from 3 separate experiments. F IL6, F (2,28) = 28.975, P < 0.0001 by two-way ANOVA; Shapiro–Wilk P = 0.5052; Tukey’s pairwise analysis: 24 hpi: 200,000 PFU vs 12,500 PFU: P < 0.0001; 200,000 PFU vs vehicle: P < 0.0001; 12,500 PFU vs vehicle: P = 0.9993; 72 hpi: 200,000 PFU vs 12,500 PFU: P = 0.9975; 200,000 PFU vs vehicle: P = 0.9914; 12,500 PFU vs vehicle: P > 0.9999; d s @ 24 hpi = 4.15; results from 3 separate experiments. Scale bar in C is 200 μm and refers to B ; scale bar in i is 100 μm and refers to i

Article Snippet: Levels of CCL2, TNFα, and IL6 were measured using a mouse inflammatory cytokine cytometric bead array multiplex panel (#552364, BD Biosciences).

Techniques: Immunostaining

Journal: Oncoimmunology

Article Title: Targeting cytokine signaling checkpoint CIS activates NK cells to protect from tumor initiation and metastasis

doi: 10.1080/2162402X.2016.1267892

Figure Lengend Snippet: Cish−/− mice respond as WT mice to LPS challenge. Groups of five B6.WT (Cish+/+) and B6.Cish−/− mice were injected i.p. with 0.2, 0.5, or 1 mg LPS/30 g mouse as indicated. (A) The weight loss and recovery upon injection of low dose LPS and (B) the survival of B6.WT and B6.Cish−/− mice upon the injection of high dose LPS was similar. Shown is the mean ± SEM of five mice per group. (C) Six hours after the injection of 0.2 mg LPS/30 g mouse, spleens were analyzed for the presence (fraction and absolute numbers) and activation (expression of CD122, CD69, and IFN-γ) of NK cells by flow cytometry (gated on live CD45+TCRβ−NK1.1+DX5+NKp46+). Naive B6.WT (Cish+/+) and B6.Cish−/− mice were used as untreated control. Shown is the summary of two independent experiments represented by the median ± interquartile range of four to six naïve and 10 LPS-challenged mice per group. Statistically significant differences between WT and Cish−/− groups as indicated were determined by one-way ANOVA with the Tukey post-test (*p < 0.05; **p < 0.01; ****p < 0.0001). (D) Serum cytokine levels were determined 7 d before (time point 0), and 1, 3, and 6 h after i.p. injection of 0.2 mg LPS/30 g mouse. Shown is the summary of two independent experiments represented by the mean ± SEM of 10 mice per group.

Article Snippet: Serum cytokine levels (G-CSF, IFN-γ, IL-1β, IL-6, IL-10, KC, MIP-1β, RANTES, and TNF-α) were determined with a CBA flex set multiplex cytokine bead array (BD Biosciences).

Techniques: Injection, Activation Assay, Expressing, Flow Cytometry

Journal: Oncoimmunology

Article Title: Targeting cytokine signaling checkpoint CIS activates NK cells to protect from tumor initiation and metastasis

doi: 10.1080/2162402X.2016.1267892

Figure Lengend Snippet: Cish−/− mice respond similarly to WT mice to CpG challenge. Groups of 5 B6.WT (Cish+/+) and B6.Cish−/− mice were injected i.p. with 0.1 mg CpG/30 g mouse. (A) Six hours after the injection of CpG, spleens were analyzed for the presence (fraction and absolute numbers) and activation (expression of CD122, CD69, and IFN-γ) of NK cells by flow cytometry (gated on live CD45+TCRβ−NK1.1+DX5+NKp46+). Three naïve B6.WT (Cish+/+) and B6.Cish−/− mice were used as untreated control. Shown is the summary of two independent experiments represented by the median ± interquartile range of 10 mice per group. Statistically significant differences between WT and Cish−/− groups as indicated were determined by one-way ANOVA with the Tukey post-test (*p < 0.05; **p < 0.01; ***p < 0.001). (B) Serum cytokine levels were determined 10 d before (time point 0), and 1, 3, and 6 h after the i.p. injection of CpG. Shown is the summary of two independent experiments represented by the mean ± SEM of 10 mice per group. Statistically significant differences between WT and Cish−/− groups as indicated were determined by the Mann–Whitney U test (*p < 0.05).

Article Snippet: Serum cytokine levels (G-CSF, IFN-γ, IL-1β, IL-6, IL-10, KC, MIP-1β, RANTES, and TNF-α) were determined with a CBA flex set multiplex cytokine bead array (BD Biosciences).

Techniques: Injection, Activation Assay, Expressing, Flow Cytometry, MANN-WHITNEY

Journal: Oncoimmunology

Article Title: Targeting cytokine signaling checkpoint CIS activates NK cells to protect from tumor initiation and metastasis

doi: 10.1080/2162402X.2016.1267892

Figure Lengend Snippet: Combining Cish deficiency and checkpoint inhibitors or cytokine stimulation shows an improved anti-metastatic effect. (A) Groups of 5–6 B6.WT (Cish+/+) or B6.Cish−/− mice were injected i.v. with 2 × 105 B16F10 melanoma cells and treated with either control Ig (cIg) (250 μg i.p. on days 0, 3, and 6), anti-PD-1 (250 μg i.p. on days 0, 3, and 6), mouse IFN-αβ (25 μg i.p. on days 0, 1, 2, and 3) or recombinant IL-2 (10,000 IU i.p. on days 0, 1, 2, and 3). (B) Groups of 5–11 B6.WT (Cish+/+) or B6.Cish−/− mice were injected i.v. with 7.5 × 105 B16F10 melanoma cells and treated with either cIg (250 μg i.p. on days 0, 3, and 6), anti-PD1/anti-CTLA-4 combination (250 μg i.p. each on days 0, 3, and 6), or recombinant IL-2 (10,000 IU i.p. on days 0, 1, 2, 3, and 4). (C) Groups of 7–10 B6.WT (Cish+/+) and B6.Cish−/− mice were injected i.p. with 1 × 105 parental RMA-S cells and treated with either PBS or recombinant IL-2 (100,000 IU i.p. on days 0, 1, 2, 3, and 4). Mice were monitored for tumor development and were euthanized at the point of abdominal swelling and discomfort. Improved survival between groups was assessed by the Log-rank Mantel–Cox test. (D) Groups of 5–15 B6.WT (Cish+/+) or B6.Cish−/− mice were injected i.v. with 5×105 RM-1 prostate carcinoma cells and treated with either cIg, anti-CD96, anti-PD-1, anti-CTLA-4, or anti-PD1/anti-CTLA-4 combination (250 μg i.p. each on days 0 and 3). (E) Groups of 8–10 B6.WT (Cish+/+) or B6.Cish−/− mice were injected i.v. with 7.5×105 B16F10 melanoma cells and treated with either 250 μg cIg or anti-CD96 mAb (i.p. on days 0 and 3). (F) Groups of 5–6 B6.WT (Cish+/+) or B6.Cish−/− mice were injected i.v. with 7.5 × 105 LWT1 melanoma cells and treated with either vehicle or PLX4720 (10 mg/kg i.p., daily from days 0 to 6). Lungs were harvested on (A, D, F) day 14 or (B, E) day 13 and macrometastases counted. Individual mice are shown by each symbol and the results are plotted as mean ± SEM. Statistically significant differences as indicated were determined by one-way ANOVA with the Tukey post-test (for multiple comparisons) (*p < 0.5; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Article Snippet: Serum cytokine levels (G-CSF, IFN-γ, IL-1β, IL-6, IL-10, KC, MIP-1β, RANTES, and TNF-α) were determined with a CBA flex set multiplex cytokine bead array (BD Biosciences).

Techniques: Injection, Recombinant