Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 1. DNA vaccination-based anti-IL-27 Abs are highly specific. The Western blot shows that our DNA vaccination-based anti-IL-27 Ab binds mouse IL-27 p28 (lane 1; 27 kDa), but not recombinant mouse IL-18, IL-12, or TNF- (lanes 2, 3, and 4, respectively). A, Coomassie Blue staining verifies the appearance of each cytokine on the loaded gel. B, Western blot showing that of these cytokines, our DNA vaccination-based anti-IL-27 Ab binds only mouse IL-27 p28. These Ab also bound natural mouse IL-27 (verified by sequencing) from supernatant of activated MOGp35–55-specific cultured primary draining lymph node cells (not shown).

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques: Western Blot, Recombinant, Staining, Sequencing, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 2. Anti-p28 Abs suppresses ongoing severe EAE. A, Four groups of 10 mice each were subjected to MOGp35–55-induced EAE. Beginning at the onset of disease (day 17), these mice were repeatedly (every other day) administered 100 g/mouse of anti-p28 Ab (f), IgG obtained from naive Lewis rats (Œ), or PBS (E). An observer blind to the experimental procedure scored EAE daily. The experiment summarized in Fig. 2 shows the results of one of three experiments performed under similar experimental conditions, with similar results. The mean maximal score SE represents six mice per group. The other four mice were killed on day 30 and subjected to histological evaluation (see Fig. 3). B, Five groups of six mice each were subjected to MOGp35–55-induced EAE. Beginning at the onset of disease (day 17), these mice were repeatedly (every other day) administered 100 g of anti-p28 Ab/mouse (f), anti-IL-18 Ab (F), anti-IL-1 Ab (), IgG obtained from Lewis rats previously subjected to an empty plasmid administration (Œ), or PBS (E). An observer blind to the experimental procedure scored EAE daily. Results are shown as the mean maximal score SE of six mice per group. C, Three groups of six mice each were subjected to induction of transferred EAE. Beginning at the onset of disease (day 5), these mice were repeatedly (every other day) administered 100 g of anti-p28 Ab/mouse (f), IgG obtained from naive Lewis rats (Œ), or PBS (E). An observer blind to the experimental procedure scored EAE daily. Results are shown as mean maximal score SE of six mice per group.

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques: Plasmid Preparation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 4. The beneficial effect of anti-IL-27 is dependent on the con- tinuing administration of protective Abs. Three groups of six mice each were subjected to active induction of EAE. Beginning 1 day after the onset of disease (day 17), these mice were treated with either a single dose of anti-p28 Ab (100 g/mouse; E) or with repeated administration (every other day) of this Ab (Œ) or PBS (f). An observer blind to the experi- mental procedure scored EAE daily. Results are shown as the mean max- imal score SE of six mice per group.

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 3. Anti-IL-27 therapy reduces the histological score of EAE. Histological evaluation was conducted 30 days after disease induction. Lumbar spinal cord samples from naive mice or from EAE mice treated with PBS, IgG from naive mice, or anti-IL-27 p28 Abs were subjected to histological analysis (nine sections each group). The arrowheads point to the parenchymal mononuclear cell infiltration. The scale for mononuclear cell infiltration used was: 0, no mononuclear cell infiltration; 1, one to five perivascular lesions per section with minimal parenchymal infiltration; 2, five to 10 perivascular lesions per section with parenchymal infiltration; and 3, 10 perivascular lesions per section with extensive parenchymal infiltration. The mean histological score SE was calculated for each group.

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 5. Protective administration of anti-IL-27 Abs decreases in vivo polarization of CD4 T cells into Th1 and suppresses IFN- production by Ag-specific T cells. C57BL/6 mice (three per group) were subjected to active induction of EAE and then to repeated administration (days 12, 14, and 16) of anti-IL-27 p28 Abs (100 g), PBS, or normal rat IgG. On day 17 cervical lymph node cells (that drain the autoimmune site) were subjected to intracellular staining of IL-4 and IFN-. A, FACS analysis of CD4 T cells in this experiment. This experiment represents results obtained in three different independent experiments with very similar data. Subsequently, cervical lymph node T cells from these mice were cultured in the presence of 100 M MOGp35–55. After 72 h of stimulation, supernatants were assayed for the protein level of IFN- (B) and IL-4 (not shown). This experiment represents results obtained in three different independent experiments with very similar data.

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques: In Vivo, Staining, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 6. Neutralizing the function of IL-27 reduces IFN- produc- tion by IFN--producing T cells. A, C57BL/6 mice (three per group) were subjected to active induction of EAE and then to repeated administration (days 3 and 6) of 100 g of anti-IL-27 p28 Abs (group 3), PBS (group 2), or normal rat IgG (group 1). On day 9, spleen cells were subjected to spot ELISA as previously described (38). A, Relative number of positive spots per 107 cultured cells. The average size of positive spots was analyzed. B, The MOGp33–55-specific CD4 T cell line was cultured with or without 100 M MOGp33–55. Cultured cells were supplemented with anti-IL-27 Abs at a final concentration of 10 g/ml (), normal rat IgG (f), or PBS (E). After 60 h of incubation, cells were plates in spot ELISA plates for an additional 24 h for the detection of IFN--positive spots (38). Number of positive spots (y-axis) and spot sizes (x-axis; logarithmic scale) determined as previously described (46).

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Concentration Assay, Incubation

Journal: Molecular Metabolism

Article Title: Elevating adipose eosinophils in obese mice to physiologically normal levels does not rescue metabolic impairments

doi: 10.1016/j.molmet.2017.12.004

Figure Lengend Snippet: Eosinophil levels in eAT, blood, bone marrow, liver, and intestine of rIL5 injected mice . (A ) Male C57BL/6J mice were fed either chow or HFD for 8 weeks and simultaneously IP injected twice weekly with rIL5 protein. (B) Flow cytometry gating strategy for eosinophils (CD45 + , live, F4/80 lo , CD11b lo , SiglecF + ) and macrophages (CD45 + , live, F4/80 hi , CD11b hi , SiglecF − ). (C) Percent eAT eosinophils negatively correlate with body weight (slope = −0.96, R 2 = 0.75). (D) Treatment with rIL5 elevates eAT eosinophils in HFD-fed mice, compared to saline and vehicle controls, back to chow-fed levels [n = 4–11]. (E) No difference in percent eosinophils in blood, bone marrow, or liver of rIL5 treated mice, but intestine does show an increase [n = 2–8]. Data are shown as means ± SEM. eAT = epididymal adipose tissue; HFD = high fat diet. Key: chow = hashed bars; HFD = open bars; saline = white; vehicle = grey; rIL5 = black. * P < 0.05, compared to saline; *** P < 0.0005, compared to saline; ˆˆ P < 0.005, compared to vehicle.

Article Snippet: During the entire 8-week period, mice also received 50 ng recombinant IL5 (rIL5; R&D Systems, Minneapolis, MN) protein (in 0.001% BSA as carrier protein) by intraperitoneal (IP) injection twice per week to elevate eosinophils.

Techniques: Injection, Flow Cytometry, Saline

Journal: Molecular Metabolism

Article Title: Elevating adipose eosinophils in obese mice to physiologically normal levels does not rescue metabolic impairments

doi: 10.1016/j.molmet.2017.12.004

Figure Lengend Snippet: Inflammatory profile of obese eAT with elevated eosinophils . (A ) Representative 40× images of eAT from HFD-fed obese mice treated with saline, vehicle, or rIL5. Macrophages (red, F4/80) are seen in abundance in all groups, whereas eosinophils (green, SiglecF) are increased in rIL5 treated mice. Magnified view shows the donut-shaped nucleus ( * ) of eosinophils juxtaposed to the spherical nucleus (#) of macrophages [n = 3]. (B) Treatment with rIL5 does not alter percent eAT macrophages or (C) protein expression of pro-inflammatory marker MHCII [n = 6–11]. (D) Gene expression analysis of macrophage marker, Adgre1 (F4/80), and an array of macrophage polarization genes were not statistically different between groups in eAT [n = 6–8]. Data are shown as means ± SEM. eAT = epididymal adipose tissue, HFD = high fat diet. Key: saline = white; vehicle = grey; rIL5 = black.

Article Snippet: During the entire 8-week period, mice also received 50 ng recombinant IL5 (rIL5; R&D Systems, Minneapolis, MN) protein (in 0.001% BSA as carrier protein) by intraperitoneal (IP) injection twice per week to elevate eosinophils.

Techniques: Saline, Expressing, Marker, Gene Expression

Journal: Molecular Metabolism

Article Title: Elevating adipose eosinophils in obese mice to physiologically normal levels does not rescue metabolic impairments

doi: 10.1016/j.molmet.2017.12.004

Figure Lengend Snippet: Weight gain, body composition, and glucose tolerance in mice with elevated AT eosinophils . (A) HFD-fed mice gained more weight over 8 weeks compared to chow, with no difference in (B) total weight gained between vehicle or rIL5 treated mice [n = 4]. HFD-fed vehicle and rIL5 treated mice have equally altered total fat and lean mass by (C) percent or (D) grams at study completion [n = 4–12]. (E) HFD-fed mice have increased body, eAT, and sAT weight, but no further difference with rIL5 [n = 4]. (F) Glucose tolerance is impaired by HFD-feeding, but not rescued by rIL5 treatment [n = 4]. Data are shown as means ± SEM. AT = adipose tissue; HFD = high fat diet; eAT = epididymal AT; sAT = subcutaneous AT. Key: chow = hashed bars, dotted lines; HFD = open bars, solid lines; vehicle = grey; rIL5 = black. * P < 0.05, compared to chow; ** P < 0.005, compared to chow; *** P < 0.0005, compared to chow; **** P < 0.0001, compared to chow; ˆ P < 0.005, compared to vehicle.

Article Snippet: During the entire 8-week period, mice also received 50 ng recombinant IL5 (rIL5; R&D Systems, Minneapolis, MN) protein (in 0.001% BSA as carrier protein) by intraperitoneal (IP) injection twice per week to elevate eosinophils.

Techniques:

Journal: Molecular Metabolism

Article Title: Elevating adipose eosinophils in obese mice to physiologically normal levels does not rescue metabolic impairments

doi: 10.1016/j.molmet.2017.12.004

Figure Lengend Snippet: Triglyceride tolerance in mice with elevated AT eosinophils . Fasted HFD-fed mice treated with vehicle or rIL5 for 8 weeks were given a bolus of TGs (i.e. olive oil) and showed no difference between groups in (A) plasma TG [n = 8] (B) plasma FFA [n = 4] (C) blood glucose [n = 8] (D) or plasma cholesterol [n = 8] over the course of 5 h. Data are shown as means ± SEM. HFD = high fat diet; TG = triglycerides; FFA = free fatty acids. Key: vehicle = grey; rIL5 = black.

Article Snippet: During the entire 8-week period, mice also received 50 ng recombinant IL5 (rIL5; R&D Systems, Minneapolis, MN) protein (in 0.001% BSA as carrier protein) by intraperitoneal (IP) injection twice per week to elevate eosinophils.

Techniques: Clinical Proteomics

Journal: Molecular Metabolism

Article Title: Elevating adipose eosinophils in obese mice to physiologically normal levels does not rescue metabolic impairments

doi: 10.1016/j.molmet.2017.12.004

Figure Lengend Snippet: Metabolic parameters of rIL5 treated mice during a mixed-meal test . HFD-fed mice treated with vehicle or rIL5 for 8 weeks were fasted and then allowed access to HFD ad libitum for 3 h. (A) Both groups consumed the same amount of food [n = 6–7]. (B) A spike in blood glucose occurred at 0.5 h following refeeding with no difference between groups [n = 6–7]. (C) Insulin also rose at 0.5 h and then held relatively steady in both groups [n = 6–7]. Plasma (D) TG [n = 6–7], (E) FFA [n = 4], and (F) cholesterol [n = 5–7] did not vary between groups upon refeeding. Data are shown as means ± SEM. HFD = high fat diet; TG = triglycerides; FFA = free fatty acids. Key: vehicle = grey; rIL5 = black.

Article Snippet: During the entire 8-week period, mice also received 50 ng recombinant IL5 (rIL5; R&D Systems, Minneapolis, MN) protein (in 0.001% BSA as carrier protein) by intraperitoneal (IP) injection twice per week to elevate eosinophils.

Techniques: Clinical Proteomics

Journal: Molecular Metabolism

Article Title: Elevating adipose eosinophils in obese mice to physiologically normal levels does not rescue metabolic impairments

doi: 10.1016/j.molmet.2017.12.004

Figure Lengend Snippet: Insulin signaling in eAT with elevated eosinophils . (A) Representative western blots of eAT showed an increase in the ratio pAKT/AKT upon insulin stimulation in HFD-fed mice that received vehicle or rIL5 for 8 weeks [n = 3–4]. β-Actin is shown as a protein loading control. (B) Quantification of western blots revealed no difference in the eAT insulin signaling response via pAKT/AKT between vehicle and rIL5 treated mice. Data are shown as means ± SEM. eAT = epididymal adipose tissue; HFD = high fat diet. Key: vehicle = grey; rIL5 = black; insulin injected = dotted bars; no-insulin saline control injection = open bars. * P < 0.05, compared to no insulin stimulation.

Article Snippet: During the entire 8-week period, mice also received 50 ng recombinant IL5 (rIL5; R&D Systems, Minneapolis, MN) protein (in 0.001% BSA as carrier protein) by intraperitoneal (IP) injection twice per week to elevate eosinophils.

Techniques: Western Blot, Control, Injection, Saline

Journal: Molecular Metabolism

Article Title: Elevating adipose eosinophils in obese mice to physiologically normal levels does not rescue metabolic impairments

doi: 10.1016/j.molmet.2017.12.004

Figure Lengend Snippet: Cold challenge: Energy balance in mice with elevated AT eosinophils . HFD-fed mice that previously received vehicle or rIL5 for 8 weeks were individually housed in metabolic cages to measure an array of physiological parameters for 2 days at RT and 2 days at 4 °C [n = 8]. (A) Average daily body weight did not vary between vehicle and rIL5 treated mice. (B) Average daily food intake and (C) average daily movement were increased in the dark cycle compared to light cycle, but with no difference between vehicle and rIL5. (D) The respiratory quotient oscillated between light and dark cycles to equal degrees in vehicle and rIL5 treated mice, both during RT and the 4 °C cold challenge; indicating both groups had the same energy substrate utilization. (E) Energy expenditure increased upon 4 °C cold challenge to maintain body heat, but did not vary between vehicle and rIL5 treated mice. Both treatment groups lost the same amount of (F) body mass, (G) fat mass, and (H) lean mass. Data are shown as means ± SEM. eAT = epididymal adipose tissue; sAT = subcutaneous AT. RT = room temperature, set at 21 °C; HFD = high fat diet. Key: vehicle = grey bars/lines; rIL5 = black bars/lines.

Article Snippet: During the entire 8-week period, mice also received 50 ng recombinant IL5 (rIL5; R&D Systems, Minneapolis, MN) protein (in 0.001% BSA as carrier protein) by intraperitoneal (IP) injection twice per week to elevate eosinophils.

Techniques:

Journal: Molecular Metabolism

Article Title: Elevating adipose eosinophils in obese mice to physiologically normal levels does not rescue metabolic impairments

doi: 10.1016/j.molmet.2017.12.004

Figure Lengend Snippet: Cold challenge: Beiging capacity of white AT with elevated eosinophils . AT of HFD-fed mice that received vehicle or rIL5 for 8 weeks was examined after 2 days of RT or 4 °C cold exposure. After cold exposure, the presence of increased eosinophils was verified in (A) sAT and (C) eAT of rIL5 treated mice compared to vehicle controls [n = 5–7]. Cold exposure alone was able to increase eosinophils in sAT but not eAT. (B) Ucp1 gene expression in sAT was increased ∼307-fold in the cold compared to RT, with no further increase in rIL5 treated mice [n = 4–5]. (D) Ucp1 gene expression in eAT trended towards an increase under cold conditions, but no difference was observed between vehicle and rIL5 treated mice [n = 5–7]. Data are shown as means ± SEM. eAT = epididymal adipose tissue; sAT = subcutaneous AT; RT = room temperature, set at 21 °C; HFD = high fat diet; Ucp1 = uncoupling protein 1. Key: vehicle = grey bars; rIL5 = black bars.

Article Snippet: During the entire 8-week period, mice also received 50 ng recombinant IL5 (rIL5; R&D Systems, Minneapolis, MN) protein (in 0.001% BSA as carrier protein) by intraperitoneal (IP) injection twice per week to elevate eosinophils.

Techniques: Gene Expression

Journal: Journal of Neuroscience

Article Title: System xc Activity and Astrocytes Are Necessary for Interleukin-1 -Mediated Hypoxic Neuronal Injury

doi: 10.1523/jneurosci.2459-07.2007

Figure Lengend Snippet: Figure 2. Role of IL-1RI signaling in the potentiation of hypoxic neuronal injury by IL-1 in vitro. A, B, Mixed cortical cell cultures were treated with 1 ng/ml IL-1 for 20–24 h in the presenceorabsenceofrIL-1ra(10–1000ng/ml;A)oranti-IL-1RI(0.1–100g/ml;B),washed, andthendeprivedofoxygen(5h).Thepercentageoftotalneuronalcelldeathwasdetermined 20–24hlater.Anasteriskindicatesvaluessignificantlygreaterthanhypoxiaalone,whereas# denotes a significant diminution of the IL-1-mediated increase in injury (IL-1) as deter- mined by one-way ANOVA followed by a Student–Newman–Keuls t test. Significance was assessed at p 0.05 (n 3–9 cultures pooled from 2–3 different experiments).

Article Snippet: In experiments using recombinant mouse IL-1ra (rIL-1ra) (R & D Systems) or monoclonal anti-mouse IL-1RI antibody directed against the extracellular domain of mouse IL-1RI (MAB7711; R & D Systems), the respective solutions were prepared with IL-1 (1.5 ng/ml; 1.5 ) and added as described above.

Techniques: In Vitro