Research ArticleInflammation

Treating murine inflammatory diseases with an anti-erythrocyte antibody

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Science Translational Medicine  21 Aug 2019:
Vol. 11, Issue 506, eaau8217
DOI: 10.1126/scitranslmed.aau8217
  • Fig. 1 The monoclonal RBC-specific antibody Ter119, an antibody with inflammatory properties, can treat murine ITP chronologically disparate from anemia.

    (A) CD-1 mice were assessed for rectal temperature (t = Pre), warmed for 5 min, and then injected with 40 μg of control rat IgG (n = 6 mice; squares) or Ter119 (n = 6 mice; circles), and rectal temperature was taken 10, 20, 30, 60, and 120 min after antibody injection. (B) Groups of CD-1 mice (n = 5 per data point) were independently assessed for anemia with terminal bleeds. Mice were treated with 40 μg of rat IgG or Ter119 and analyzed at the time points indicated. The time point represented as zero is a prebleed for the mice treated with IgG or Ter119. (C) To assess the ability of Ter119 versus control IgG to increase platelet counts in a passive ITP model over time, mice were first treated with Ter119 or IgG for the duration depicted on the x axis and terminally bled for platelet counts. Sixty minutes before this, terminal bleed mice were injected with 3 μg of antiplatelet antibody to induce thrombocytopenia. For clarity, the Ter119-treated mice at the 1.5-hour time point in (C) were injected with Ter119 (intravenously) for 30 min followed by antiplatelet antibody (intravenously) and then bled for platelet counts after a further 60 min. The time point represented as zero in (B) is a prebleed for all mice in the experiment. Data are presented as means ± SEM from five separate experiments with one mouse for each data point in each experiment (80 mice in total for the subpanel); each time point (except for time 0) is a terminal bleed and assesses different mice. Statistical analysis was performed using multiple t tests. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 in comparison to the same control IgG time point.

  • Fig. 2 Ter119 inhibits inflammatory arthritis in the K/BxN model.

    C57BL/6 mice were assessed for basal arthritis measurements on day 0 (A and B), and then, one group of mice received 45 μg of Ter119 (circles), the other group (squares) received nothing. Two hours later, all mice received an injection of K/BxN serum. Ankle measurements (A) and clinical score (B) were taken every day for 10 days. Data are expressed as means ± SEM divided across four separate experiments with ≥7 mice per group. Mice received an injection of K/BxN serum with no pretreatment (C and D). On day 5, arthritic mice were treated (arrow) with nothing (squares), 50 μg of control anti-RBC antibody 30-F1 (triangles), or 45 μg of Ter119 (circles). Ankle measurements (C) and clinical score (D) were measured on days 0 to 2 and days 5 to 9. Data are expressed as means ± SEM divided across four separate experiments. n = 5 (K/BxN serum alone); n = 6 (Ter119); n = 7 (30-F1). To assess the requirement for Fc region glycosylation in the therapeutic function of Ter119, C57BL/6 mice were injected with K/BxN serum for 5 days and then administered with either no treatment (squares), 45 μg of Ter119 (circles), or 45 μg of Ter119 that had been previously deglycosylated using PNGase F (triangles), and ankle measurements (E) and clinical score (F) were assessed. n = 7 (K/BxN serum alone); n = 7 (Ter119); n = 8 (degly-Ter119). Statistical analysis was performed using multiple t tests (A and B) and two-way analysis of variance (ANOVA) test with Dunnett’s multiple comparisons test compared to the control arthritis group (no treatment group) (C to F). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 3 Ter119 inhibits the progression of inflammatory arthritis in the CAIA model.

    (A) C57BL/6 mice injected with the collagen antibody cocktail (day 0) and LPS (day 3) were allowed to develop arthritis and then injected (day 5) with either Ter119 or an IgG2b isotype control antibody (2 mg/kg), and the clinical scores were evaluated until day 12. (B) Representative paws from mice on day 8 of the experiment are shown. The mice were assessed for histological score on day 12. Representative histology from the paws of mice therapeutically treated with the IgG2b isotype control antibody (C) or Ter119 (D) are shown. JS, joint space; AC, articular cartilage; S, synovium; B, bone. (E) The histological scores out of 15 for individual mice are shown with two sections taken per paw and one paw per mouse, always the rear right ankle cut at similar midsection depth. ***P < 0.001, Mann-Whitney two-tailed test.

  • Fig. 4 Ter119 induces dose-dependent changes in CAIA and monocyte surface receptors.

    (A) C57BL/6 mice (10 per dosage group) were injected with the collagen antibody cocktail (day 0) and LPS (day 3), allowed to develop arthritis for 5 days, and injected (arrow) with the IgG2b isotype control antibody (2 mg/kg; Ctrl, square) or Ter119 (1, 1.5, or 2 mg/kg). Mean clinical scores for treatment period (days 6 to 12) (essentially, the area under the curve): 1.0 mg/kg (not significant), 1.5 mg/kg (P < 0.05), 2.0 mg/kg (P < 0.001), compared to the control; Kruskal-Wallis with Dunn’s multiple comparisons test. (B) Mice were evaluated for histological score per mouse (maximum score, 36 per mouse) on day 12 for each dosage. One day after the isotype control or Ter119 injection (day 6), four mice from each group were used to assess the parameters displayed in (C), and the remaining mice allowed to develop arthritis for the 12 day duration of the experiment. A single-cell suspension for each mouse was made from the spleen, and monocyte FcγRI (CD64), FcγRIIB/IIIA (CD16/32), CR1/2 (CD21/35), and C5aR (CD88) mean fluorescence intensities (MFIs) were quantitated by flow cytometry (C). Naïve mice were not induced to develop arthritis. One-way ANOVA test with Dunn’s multiple comparison as compared to the control arthritis group. Not significant (ns),*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 5 Ter119 induces changes in chemokines and cellular migration in CAIA.

    (A) The same mice that were injected with the collagen antibody cocktail (day 0) and LPS (day 3) in Fig. 4 were used to assess the parameters in this figure 1 day after Ter119 or isotype control injection (day 6). Naïve mice were unmanipulated. To assess plasma chemokines CCL2, CXCL10, CXCL5, CXCL9, CCL5, and CCL11 (Eotaxin-1), plasma from each mouse was collected 1 day after isotype control or Ter119 injection (day 6 of the experiment) and chemokines were assessed by Luminex. (B) To evaluate changes in monocyte percentages in the blood, spleen, and liver, single-cell suspensions were made and monocytes were enumerated by flow cytometry. (C) To assess the number of infiltrating cells in the joints, the patellas from each mouse were collected and digested, and infiltrating leukocytes were enumerated by visual count. (D) To assess chemokines and cytokines from the joints, synovial fluid from each mouse was assessed by Luminex. One-way ANOVA test with Dunn’s multiple comparison as compared to the control arthritis group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 6 Ter119 but not deglycosylated TER119 ameliorates CAIA.

    (A and B) Arthritis was induced in 12 mice per group on day 0 followed by LPS on day 3, and all 12 mice from each group were injected with either phosphate-buffered saline (PBS; squares), Ter119 (1.5 mg/kg; circles), or deglycosylated Ter119 (1.5 mg/kg; triangles) on day 5, and clinical scores were assessed. Erythrocyte sensitization was evaluated on day 6 (C) and day 8 (D) by staining the ex vivo erythrocytes with anti-rat IgG-PE, and MFIs of the erythrocytes were assessed. Erythrocytes were enumerated from the blood by a MACS Quant flow cytometer (E) with individual mice and statistics shown in fig. S3. Four mice from each group were sacrificed on day 6 to assess inflammatory cell infiltration from the patellas (F) and assess CCL2 concentrations from the blood (G) and from the synovial fluid (H). Statistics are as follows: (B) Kruskal-Wallis with Dunn’s test; (C, D, and F to H) one-way ANOVA with Tukey’s multiple comparison test. *P < 0.05, **P < 0.01, and ****P < 0.0001.

  • Fig. 7 Ter119 expressed as murine IgG switch variants can treat a chronic model of CIA independent of passive-antibody transfer.

    DBA/1 mice immunized against chick type II collagen were allowed to develop arthritis and then treated (timing as denoted by the arrow) with PBS (n = 7 mice; square) and Ter119 (2 mg/kg) expressed as a murine IgG1subtype (n = 6 mice; gray triangles) or expressed as a murine IgG2a subtype (n = 6 mice; inverted triangles), and arthritis clinical score was evaluated over the course of the experiment. Statistical comparison was performed using two-way ANOVA for repeated measures (assuming normality) with Tukey’s multiple comparisons test. *P < 0.05 and **P < 0.01 (gray, PBS versus IgG1; black, PBS versus IgG2a).

  • Fig. 8 Ter119 inhibits TRALI.

    (A) SCID mice were injected with 40 μg of Ter119 (circles and triangles) or left untreated (squares) for 24 hours. Mice were then injected with 50 μg of 34-1-2s (triangles and squares) or nothing (circles). Rectal temperatures were measured every 30 min for 2 hours after the second injection (A), and mice were subsequently sacrificed at 2 hours to assess pulmonary edema (B) and pulmonary neutrophil accumulation (C). Data are expressed as means ± SEM from four separate experiments. n = 4 (Ter119); n = 5 (34-1-2s); n = 14 (Ter119 + 34-1-2s). Statistical comparisons were made after testing for normality. A two-way ANOVA with Tukey’s multiple comparison testing was performed in (A), a Kruskal-Wallis test with Dunn’s multiple comparison testing was performed for (B), and a one-way ANOVA with Tukey’s multiple comparison testing was performed for (C). *P < 0.05, **P < 0.01, ***P < 0.001, and **** P < 0.0001.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/506/eaau8217/DC1

    Fig. S1. Priming with Ter119 can protect mice from Ter119-induced hypothermia.

    Fig. S2. Ter119 substantially reduces the detection of complement components in the joint of arthritic mice.

    Fig. S3. Ter119 and deglycosylated Ter119 induce anemia over time in the CAIA model.

    Fig. S4. Ter119 class-switched to murine IgG1 and murine IgG2a sequences.

    Data file S1. Primary data.

  • The PDF file includes:

    • Fig. S1. Priming with Ter119 can protect mice from Ter119-induced hypothermia.
    • Fig. S2. Ter119 substantially reduces the detection of complement components in the joint of arthritic mice.
    • Fig. S3. Ter119 and deglycosylated Ter119 induce anemia over time in the CAIA model.
    • Fig. S4. Ter119 class-switched to murine IgG1 and murine IgG2a sequences

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    Other Supplementary Material for this manuscript includes the following:

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