Neutrophil Responses to Sterile Implant Materials

Abstract

In vivo implantation of sterile materials and devices results in a foreign body immune response leading to fibrosis of implanted material. Neutrophils, one of the first immune cells to be recruited to implantation sites, have been suggested to contribute to the establishment of the inflammatory microenvironment that initiates the fibrotic response. However, the precise numbers and roles of neutrophils in response to implanted devices remains unclear. Using a mouse model of peritoneal microcapsule implantation, we show 30-500 fold increased neutrophil presence in the peritoneal exudates in response to implants. We demonstrate that these neutrophils secrete increased amounts of a variety of inflammatory cytokines and chemokines. Further, we observe that they participate in the foreign body response through the formation of neutrophil extracellular traps (NETs) on implant surfaces. Our results provide new insight into neutrophil function during a foreign body response to peritoneal implants which has implications for the development of biologically compatible medical devices.

Fig 1. Flow cytometry schematics.

Representative flow cytometry contour plots describing the gating scheme used to identify different immune cell subsets (isolated 2 weeks followed alginate microcapsule implantation) in the peritoneal cavity. All single cells retrieved from the peritoneal cavity were run through flow cytometry.

Fig 2. Increased neutrophil presence in peritoneal exudate following microcapsule implantation.

(A)–Representative flow cytometry contour plots showing percentages of neutrophils (CD11b⁺ Ly6G⁺) in the peritoneal exudate of mice implanted with microcapsules made of different materials. (B)–Counts of neutrophils in the peritoneal exudate 2 weeks following implantation of microcapsules made of different materials compared to control untreated and mock treated mice. C–Counts of monocyte/macrophage (CD11b⁺ Ly6G^- CD11c^-), dendritic cells (CD11b⁺ CD11c⁺), B cells (CD19⁺), and T cells (TCRβ⁺) in the peritoneal exudate 2 weeks following implantation of microcapsules made of different materials compared to control untreated and mock treated mice. Mock treatment entailed performing a laparotomy and injecting sterile saline (sham surgery). *** indicates p<0.001, using one-way ANOVA followed by Bonferroni post-test comparing specific sample to mock or untreated. Data are representative of at least 2 independent experiments with total n ≥ 5.

Fig 3. Increased neutrophil presence is due to biomaterial implants.

(A)–Comparison of neutrophil counts in mice implanted with alginate in its solution or cross-linked hydrogel microcapsule form (measured 2 weeks following implantation). Mock and alginate microcapsule datasets are the same as presented in Fig 2. (B)–Comparison of neutrophil counts in mice implanted with PLGA that will or will not degrade in 2 weeks (measured 2 weeks following implantation). Datasets on PLGA microcapsules that do not degrade are the same as presented in Fig 2. Data are based on at least 2 independent experiments with n ≥ 5 for A and B. *** indicates p<0.001, using one-way ANOVA followed by Bonferroni post-test. (C)–Comparison of neutrophil counts in mice implanted with glass microcapsules that did or did not undergo pyrolysis treatment. Data are based on at least 1 independent experiment with n ≥ 4. 'ns' indicates not significant. Datasets on glass microcapsules that were not pyrolysis treated are the same as presented in Fig 2. (D)–Dependence of number of neutrophils observed in the peritoneal exduate on the number of microcapsules implanted, and less to absence of dependence on surface area of microcapsules. Black dots represent ~500μm, green dot represents ~300μm, red dot represents ~800μm and blue dot represents ~2000μm alginate microcapsules. Data are based on at least 1 independent experiment with n ≥ 5 mice for each microcapsule count. (E)–Curve fitting for data presented in 'D'. A non-linear 3-parameter dose response curve (mean of Log[neutrophil counts] vs Log[microcapsule count] or Log[microcapsule surface area]) was fitted assuming that microcapsules act as a stimulant for neutrophils. R² value was 0.96 for the fit using the microcapsule counts, suggesting a direct correlation between neutrophil counts and microcapsule counts.

Fig 4. Neutrophil Function.

(A)–Confirmation of neutrophil phagocytic capacity. Fluorescent nanoparticles (~190 nm polystyrene nanoparticles) were injected intraperitoneally, 1 week following alginate microcapsule implantation. Left–Representative flow cytometry histograms generated following gating on Ly6G⁺ cells showing nanoparticles (NP) associated with neutrophils. Grey histograms are fluorescence intensities in control mice that have not been injected with nanoparticles. Right–Quantification of the NP uptake histograms, showing a large increase in NP MFI 3 hours post NP injection that drops over time. Data are representative of at least 1 independent experiment with total n ≥ 4. (B)–Multiplex luminex assay to measure chemokines and cytokines secreted by neutrophils. Neutrophils were isolated using a magnetic bead based negative selection technique, followed by ex vivo overnight culture. Higher amounts of key inflammatory cytokines and chemokines are secreted by peritoneal cavity but not bone marrow neutrophils. B.D.L. = below detectable levels. ** and *** indicate p<0.01 and p<0.001, respectively, using a two-tailed Student's t test with Welch's correction (for samples where the levels of cytokine/chemokine are above detectable levels). # indicates p<0.01 using a two-tailed Fisher's exact test, for samples where the levels of cytokine/chemokine were below detectable levels. Data presented are based on n = 6.

Fig 5. Neutrophil extracellular traps.

Representative z-stacked immunofluorescence images showing neutrophil elastase and DNA/histone-H1 on the surface of microcapsules. Alginate microcapsules were retrieved 1–2 weeks following implantation, while Polystyrene and PMMA microcapsules were retrieved 3 days following implantation. Images are representative of at least 2 independent experiments with total n ≥ 5 mice, and imaging of multiple retrieved microcapsules from each mouse. Scale bar = 100 μm.