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. 2023 Jun 16;9(24):eade9488.
doi: 10.1126/sciadv.ade9488. Epub 2023 Jun 16.

Identification of a humanized mouse model for functional testing of immune-mediated biomaterial foreign body response

Joshua C Doloff  1   2   3   4 , Minglin Ma  1   2 , Atieh Sadraei  1   3 , Hok Hei Tam  1   3 , Shady Farah  1   2   3 , Jennifer Hollister-Lock  5 , Arturo J Vegas  1   2 , Omid Veiseh  1   2 , Victor M Quiroz  4 , Amanda Rakoski  4 , Stephanie Aresta-DaSilva  1   2 , Andrew R Bader  1   2 , Marissa Griffin  1 , Gordon C Weir  5 , Michael A Brehm  6 , Leonard D Shultz  7 , Robert Langer  1   2   3   8   9 , Dale L Greiner  6 , Daniel G Anderson  1   2   3   8   9
Affiliations

Identification of a humanized mouse model for functional testing of immune-mediated biomaterial foreign body response

Joshua C Doloff et al. Sci Adv. .

Abstract

Biomedical devices comprise a major component of modern medicine, however immune-mediated fibrosis and rejection can limit their function over time. Here, we describe a humanized mouse model that recapitulates fibrosis following biomaterial implantation. Cellular and cytokine responses to multiple biomaterials were evaluated across different implant sites. Human innate immune macrophages were verified as essential to biomaterial rejection in this model and were capable of cross-talk with mouse fibroblasts for collagen matrix deposition. Cytokine and cytokine receptor array analysis confirmed core signaling in the fibrotic cascade. Foreign body giant cell formation, often unobserved in mice, was also prominent. Last, high-resolution microscopy coupled with multiplexed antibody capture digital profiling analysis supplied spatial resolution of rejection responses. This model enables the study of human immune cell-mediated fibrosis and interactions with implanted biomaterials and devices.

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Figures

Fig. 1.
Fig. 1.. Identifying a humanized model capable of biomaterial fibrosis.
500-μm-diameter SLG20 alginate spheres (0.5-ml volume) were implanted into the IP space of C57BL/6 and various humanized mouse models for 28 days and then analyzed for the degree of fibrosis. (A) Graphic showing steps required to achieve fibrotic FBR in the human HSC–engrafted NSG-SGM3 BLT model. BM, bone marrow; IV, intravenous. (B) Bright-field images of retrieved spheres reveal significant overgrowth in control C57BL/6 mice (left), with no fibrosis in NSG BLT mice, without or with xenogeneic neonatal pig cell clusters (NPCCs) (middle images). FBR was only observed after transgenic expression of three human cytokines [SCF, GM-CSF, and IL-3 (SGM3)] for innate immune cell maintenance. (C) Bright-field images (4× and 10×) of fibrosed spheres retrieved from engrafted NSG-SGM3 BLT mice 28 days after implantation. (D) Collagen levels on retrieved capsules by hydroxyproline assay from nonengrafted NSG-SGM3 BLT (white), wild-type C57BL/6 (gray), or engrafted (humanized) NSG-SGM3 BLT mice (red). (E) Phase contrast images of fibrosed and epididymal fat-embedded 5-mm PDMS discs and 500-μm PS spheres retrieved 28 days after IP implantations (alginate in fig. S1B). (F) Flow cytometry analysis reflected similar CD45+ engraftment without (blue) versus with (red) SGM3 incorporation in the blood, spleen, and bone marrow; however, a significant increase occurred in the IP space. PEC, peritoneal exudate cell. (G) With SGM3 adoption, CD45+CD3−CD20−CD33+ innate myeloid lineages were significantly increased in all compartments. For statistical analysis, one-way analysis of variance (ANOVA) with Bonferroni multiple comparison correction was used. *P < 0.05 and ***P < 0.0001; ns, not significant. n = 5 (biologic replicates) per group. Experiments were repeated three times.
Fig. 2.
Fig. 2.. FBR human macrophage dependence and mouse fibroblast cross-talk in NSG-SGM3 BLT humanized model.
(A) As compared to nonfibrosed (nonengrafted) and fibrosed (engrafted) controls, clodrosome depletion of human macrophages resulted in complete loss of fibrosis on 500-μm-diameter SLG20 alginate spheres following 2-week IP implantations in engrafted NSG-SGM3 BLT mice. (B) Flow cytometry analysis of human immune cell numbers (#) dissociated from capsule spheres (Caps) or PECs retrieved 2 or 4 weeks after IP implantation (green versus blue, respectively) showing significant increases of human (h)CD45+CD3−CD20−CD33+ myeloid cells, which were eliminated following clodrosome macrophage (Mϕ) depletion throughout 2-week implantations (red). By comparison, human neutrophils (CD45+CD66b+) did not significantly change. (C) As compared to very high absolute human immune cell numbers [in (B)], effective mouse immune cell elimination by irradiation was confirmed by low counts of residual mouse (m) macrophages (F4/80+CD11b+) and neutrophils (Ly6g+CD11b+), with no significant response by either population to material implantation or macrophage depletion. (D) NanoString analysis of cell and cytokine markers on alginate spheres at 1, 5, and 14 days after implantation in engrafted NSG-SGM3 BLT mice versus non-engrafted and engrafted but macrophage-depleted controls. Similar to wild-type FBR, macrophage and B cell markers were some of the largest dynamic responders. White, within background of the assay. (E) No human collagen was observed; instead, delayed mouse αSMactin fibroblast and collagen (Col1a1) expression indicated mouse myofibroblast engagement. For all, n = 5 (biologic replicates) per treatment. Flow cytometry was performed twice, and NanoString was performed once. For flow comparisons, one-way ANOVA (***P < 0.0001 versus clodrosome-depleted controls) was used. For NanoString, log2 scale was used; for statistical analysis, see Materials and Methods.
Fig. 3.
Fig. 3.. FBR and foreign body giant cell formation is observed in the SC site in engrafted NSG-SGM3 BLT humanized mice.
500-μm SLG20 alginate or PS spheres (0.5 ml) and 5-mm PDMS discs were implanted for 14 or 28 days into the SC space of NSG-SGM3 BLT humanized mice and analyzed for degree of fibrosis upon retrieval. (A) H&E- and Masson’s Trichrome–stained histological sections of excised SC tissue 14 and 28 days after implantation for alginate and 14 days for PS (additional photos in figs. S8 and S9) (4×). Scale bars, 500 μm. (B) Confocal microscopy showing prominent FBGC formation (by macrophage marker CD68, green) colocalized with an inner ring of myofibroblast marker αSMactin (red) at day 28. (C) Photos showing prominent encapsulation of alginate and PS spheres upon retrieval following 4-week SC implantations. (D) Confocal microscopy confirming that the majority of earlier identified (Figs. 1 and 2) CD33+ myeloid cells coexpress macrophage marker CD68. CD68 and CD33 antibodies are anti-human; our αSMactin Ab is pan-species and detecting mouse fibroblasts here. (E) NanoString analysis of physical cell and cytokine markers analyzed from deposited cell RNA extracts from PS spheres and 5-mm PDMS discs collected at 14 and 28 days after IP or SC implantations in engrafted NSG-SGM3 BLT mice as compared to nonengrafted controls. White, within assay background. NanoString analysis was performed once (log base-2 scale); for statistical analyses, see Materials and Methods. n = 5 (biologic replicate) mice per group for all assays. Experiments were repeated three times.
Fig. 4.
Fig. 4.. DSP of human immune presence on and around implanted biomaterials in engrafted NSG-SGM3 BLT mice.
500-μm-diameter SLG20 alginate or PS spheres (0.5 ml) were implanted into the SC space of humanized NSG-SGM3 BLT mice for 14 and 28 days. (A) Per NanoString digital spatial profiling (DSP) protocols, immunofluorescence microscopy with DAPI nuclear (blue), leukocyte CD45 (red), and macrophage CD68 (green) staining was used to identify (whole) ROIs (white lines) for UV laser ablation to decouple probes from a 30-plex (anti-human, h) Ab panel. (B) With the DSP team, we found that concentric ring ROIs could be collected around the same implant with successive rounds of ablation where the UV laser was incrementally increased to 15 μm, thereby capturing data as a function of distance from its surface. (C) Heatmaps showing the ROI data for all 30 antibodies for both 2-week and 4-week alginate and PS implants. (D) Log-scale line graphs of data [as reflected in (C)]. Muted at 2 weeks, alginate FBR is as robust as PS’s by 4 weeks. (E) On the basis of our concentric ring DSP method [in (B)], macrophage CD68 was largely localized at the implant surface, while B cell markers CD19 and CD20 decreased, slowly moving away. One-way ANOVA (Bonferroni comparison) was used (*P < 0.05 and ***P < 0.0001, versus 0-μm distance). Data are presented as means ± SE. n = 5 (biologic replicate) mice per group. DSP analysis was run once across n = 2 biologic replicates per treatment group (SLG20 and PS) per time point (2 and 4 weeks), with 20 areas of interest per sample. STAT3, signal transducers and activators of transcription 3; P-STAT3, phosphorylated STAT3; IgG, immunoglobulin G.
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