Granulocyte-macrophage colony-stimulating factor autoantibodies in murine ileitis and progressive ileal Crohn's disease

Abstract

Background & aims: Genetic variations that affect innate immunity increase risk of ileal Crohn's disease (CD). However, the penetrance of susceptibility genes, including NOD2, is low, suggesting additional risk factors. Neutralizing autoantibodies (Ab) against granulocyte-macrophage colony-stimulating factor (GM-CSF Ab) reduce neutrophil antimicrobial function in patients with primary alveolar proteinosis (PAP). We investigated whether GM-CSF Ab regulates neutrophil function in CD.

Methods: Serum samples from 354 adult and pediatric patients with inflammatory bowel disease (IBD) were analyzed for GM-CSF Ab and IBD markers. Levels of GM-CSF Ab were compared with patients' CD features and neutrophil function. Intestinal barrier function and nonsteroidal anti-inflammatory drug (NSAID)-induced injury were assessed in GM-CSF-null and NOD2-null mice.

Results: Median GM-CSF Ab levels increased from 0.4 microg/mL in control serum to 2.4 microg/mL in pediatric CD and 11.7 microg/mL in adult CD serum and were associated with ileal involvement (P<.001). Ileal location, duration of disease, and increased GM-CSF Ab levels were associated with stricturing/penetrating behavior (odds ratio, 2.2; P=.018). The positive and negative predictive values of GM-CSF Ab for stricturing/penetrating behavior were comparable with that of other IBD serum markers. CD patients with increased GM-CSF Ab had reduced neutrophil phagocytic capacity and increased accumulation of pSTAT3+ neutrophils in the affected ileum. GM-CSF-null mice and NOD2-null mice in which GM-CSF was neutralized had defects in mucosal barrier function and developed a transmural ileitis following NSAID exposure.

Conclusions: GM-CSF regulates ileal homeostasis in CD and in mouse models. CD patients with increases in serum GM-CSF Ab might benefit from GM-CSF administration.

Figure 1.. GM-CSF Ab and Crohn’s Disease Location and Behavior.

A) Serum GM-CSF autoantibody (GM-CSF Ab) concentration was determined by ELISA recognizing glycosylated GM-CSF in the pediatric (A1) and adult (A2) onset CD patients with colon-only (L2) or ileal (L1)/ileo-colonic (L3) location, UC patients and healthy controls (CTRL) summarized in Table I (n = 354 IBD and 108 controls). *p<0.001 vs. A1 CTRL, A1UC, and A1L2; **p<0.001 vs. A2 CTRL, A2UC. B & C) The relationship between the odds for B) ileal location and C) stricturing/penetrating behavior, and serum GM-CSF Ab concentration in CD is shown. Serum GM-CSF Ab was divided into subgroups by decile, and a smooth line was generated by robust locally weighted regression (LOWESS). D) Kaplan-Meier survival curve analysis was performed to determine the frequency of CD patients free from stricturing/penetrating disease behavior as a function of serum GM-CSF Ab level and duration of disease. E) The frequency of stricturing/penetrating disease behavior was determined in the groups shown. *p<0.01 vs. age & location matched GM-CSFAb-. In D & E: GM-CSF Ab+: serum concentration ≥ 1.6 mcg/ml. Data are shown as the median(range) or frequency.

Figure 2.. GM CSF Ab and IBD Serologies.

A & B) The frequency of sero-positivity for the IBD serological markers ASCA, CBir1, OmpC, I2, and ANCA was determined and is shown as a function of disease location and GM-CSF Ab level for a subset of the pediatric onset group (n=157) for which these values were obtained. *p<0.01 vs. location matched GM-CSFAb-. C) The positive and negative predictive value (PPV & NPV) of the GM-CSF Ab assay and the ASCA, ANCA, OmpC, and I2 markers for predicting progression to structuring/penetrating behavior was determined in the pediatric onset cohort. Because patients were followed for varying periods of time, we used progression-free probabilities to calculate PPV and NPV. This relationship was as shown. Each line going from left to right depicts the PPV and NPV for each biomarker as a function of time, with the earliest time point (stricturing by 12 months after diagnosis) represented by the start of the line at the left lower end, the first closed circle on the line representing 60 months after diagnosis, and the longest duration of disease (108 months) at the upper right end. GM-CSF Ab+: serum concentration ≥ 1.6 mcg/ml. Data are shown as the relative frequency.

Figure 3.. GM CSF Ab and Neutrophil Function.

A) Neutrophils were stimulated with GM-CSF (0, 1, or 10 ng/ml) in whole blood samples obtained from healthy controls, CD patients with low (GM-CSF Ab Lo) or high (GM-CSF Ab Hi) serum GM-CSF Ab, and disease controls with Primary Alveolar Proteinosis (PAP), and the mean fluorescent intensity (MFI) for cell surface CD11b was determined by flow cytometry (n = 6 per group). Data are shown as the mean (SEM), *:p<0.05 versus GM-CSF Ab Hi group at same GM-CSF dose. B) Neutrophil phagocytic capacity was determined in whole blood samples obtained from healthy controls (CTRL), CD with low (CD Lo) or high (CD Hi) serum GM-CSF Ab, and disease controls with PAP by flow cytometry (n = 6–14 per group). *:p<0.05 vs CTRL. C) The Pearson correlation between serum GM-CSF Ab and neutrophil phagocytic capacity was determined in healthy controls (CTRL) and CD patients.

Figure 4.. GM CSF Ab and Neutrophil STAT3 Activation.

A) The frequency of circulating neutrophils in healthy controls and CD patients with low (GM-CSFAb Lo) or high (GMCSFAb Hi) serum GM-CSF Ab containing tyrosine phosphorylated STAT3 (pSTAT3) was determined by intra-cellular staining for pSTAT3 and flow cytometry as shown. B) The frequency of circulating neutrophils containing pSTAT3 was determined in CD patients in remission (active-), or with clinically active disease (active+), and low or high serum GM-CSF Ab (n=5–7 per group). Data are shown as the mean (SEM), *p<0.05 vs. GM-CSF Ab Lo with inactive disease. C) Circulating neutrophils obtained from CD patients were stimulated with GM-CSF (10 ng/mL) or PBS for 30 minutes. The frequency of neutrophils containing pSTAT3 was determined by flow cytometry (n=20). *p<0.05 vs. untreated sample. D) The frequency of neutrophil elastase (NE) staining or pSTAT3+ cells was determined by immunofluoresence (IF) or immunohistochemistry in ileal sections obtained from healthy controls, or CD patients with low (GM-CSF Ab Lo) or high (GM-CSF Ab Hi) serum GM-CSF Ab. For the IF images, the NE stain (green) is shown with co-labeling for cell nuclei with DAPI (blue), or in the inset, pSTAT3 (red). Images representative of 5–7 per group are shown.

Figure 4.. GM CSF Ab and Neutrophil STAT3 Activation.

A) The frequency of circulating neutrophils in healthy controls and CD patients with low (GM-CSFAb Lo) or high (GMCSFAb Hi) serum GM-CSF Ab containing tyrosine phosphorylated STAT3 (pSTAT3) was determined by intra-cellular staining for pSTAT3 and flow cytometry as shown. B) The frequency of circulating neutrophils containing pSTAT3 was determined in CD patients in remission (active-), or with clinically active disease (active+), and low or high serum GM-CSF Ab (n=5–7 per group). Data are shown as the mean (SEM), *p<0.05 vs. GM-CSF Ab Lo with inactive disease. C) Circulating neutrophils obtained from CD patients were stimulated with GM-CSF (10 ng/mL) or PBS for 30 minutes. The frequency of neutrophils containing pSTAT3 was determined by flow cytometry (n=20). *p<0.05 vs. untreated sample. D) The frequency of neutrophil elastase (NE) staining or pSTAT3+ cells was determined by immunofluoresence (IF) or immunohistochemistry in ileal sections obtained from healthy controls, or CD patients with low (GM-CSF Ab Lo) or high (GM-CSF Ab Hi) serum GM-CSF Ab. For the IF images, the NE stain (green) is shown with co-labeling for cell nuclei with DAPI (blue), or in the inset, pSTAT3 (red). Images representative of 5–7 per group are shown.

Figure 5.. GM CSF Ab Administration and Barrier Function in Mice.

A) GM-CSF antibody (50 mcg IP) or isotype control were administered to wild type mice and serum was obtained two weeks later. The circulating level of GM-CSF Ab (mcg/mL) was determined by ELISA (n = 6 per group). *p<0.05 vs. IgG treated control. B) GM-CSF antibody (GM-CSF Ab, 50 mcg IP) or isotype control (IgG) were administered to wild type mice. Two weeks later, circulating leukocytes were obtained and stimulated with GM-CSF (0, 1, or 10 ng/mL) in whole blood samples and cell surface cd11b abundance was determined by flow cytometry. The cd11b stimulation index was calculated and is shown (n = 6 per group). *p<0.05 vs. GM-CSF Ab treated group at GM-CSF dose of 10 ng/mL. C) Ileal para-cellular permeability to FITC-dextran were determined using the everted gut sac method in mice fed regular chow, with and without GM-CSF Ab pre-treatment for two weeks as shown (n = 10–12 per group). *p<0.05 versus WT on regular chow. D) Bacterial translocation to draining mesenteric lymph nodes was determined in mice fed regular chow, with and without GM-CSF Ab pre-treatment for two weeks as shown (n = 10–12 per group). *p<0.05 versus WT on regular chow, **p<0.05 vs C15KO on regular chow. WT: wild type, GMKO: gm-csf deficient, C15KO: card15 deficient, GM-CSF Ab: received neutralizing GM-CSF antibody, 50 mcg IP. Data are shown as the A) median (range) or B-D) mean (SEM).

Figure 6.. GM-CSF Ab and Susceptibility to NSAID-Induced Ileitis.

A & B) Gm-csf deficient (GMKO), card15 deficient (C15KO) or wild type (WT) control mice were treated with a neutralizing GM-CSF antibody (GM-CSF Ab, 50 mcg IP) or isotype control and were placed on regular chow, or chow containing the NSAID piroxicam (PIR, 200 ppm) two weeks later. The effect upon A) ileal and B) colonic histopathology was determined (n = 10–12 per group). Data are shown as the mean (SEM), *p<0.05 versus WT on piroxicam, **p<0.05 vs C15KO on piroxicam. Original magnification, × 100; bar = 50 μm.