Neutrophils enhance the clearance of systemic amyloid deposits in a murine amyloidoma model

Abstract

Introduction: Amyloid-specific antibodies have been shown to opsonize and enhance amyloid clearance in systemic amyloidosis mouse models. However, the immunological mechanisms by which amyloid is removed have not been clearly defined. Previous reports from preclinical in vivo studies suggest polymorphonuclear cells (i.e., neutrophils) can affect amyloid removal. Therefore, we sought to analyze how neutrophils may contribute to the clearance of human AL amyloid extracts, using a murine amyloidoma model.

Methods: Immunocompromised nude mice injected subcutaneously with patient-derived AL amyloid extract (generating a localized "amyloidoma") were used to circumvent confounding factors contributed by the adaptive immune system and served as the model system. Two representative AL amyloid extracts were used, ALλ(CLA), which is refractory to clearance, and ALκ(TAL), which is readily cleared in mice. Neutrophil recruitment to the amyloid masses, cellular activation, and propensity to engulf amyloid were assessed.

Results: Immunophenotyping of amyloidomas from animals implanted with 2 mg of either ALλ or ALκ revealed that more neutrophils were recruited to ALκ amyloid masses as compared to the ALλ material, which was generally devoid of neutrophils. Ex vivo analyses indicated neutrophils do not efficiently phagocytose amyloid directly. However, histological evaluation of the ALκ amyloidoma revealed the abundant presence of neutrophil extracellular traps, which were absent in the ALλ amyloidomas. Using neutrophil depletion experiments in mice, we determined that mice devoid of neutrophils cleared the human amyloid lesions less efficiently. Moreover, mice devoid of neutrophils also had significantly reduced intra-amyloid expression of inflammatory cytokines.

Discussion: Neutrophils may not directly mediate amyloid clearance through phagocytosis; however, these cells can be stimulated by the amyloid and may function to facilitate phagocytosis and amyloid clearance by professional phagocytes (e.g., macrophages).

Keywords: AL amyloidosis; amyloid phagocytosis; amyloid resolution; neutrophil NETs; neutrophils in amyloid.

Figure 1

Comparison of ALλ(CLA) and ALκ(TAL) patient-derived material. Transmission electron microscopic images of indicated patient extract (A, B), black scale bars represent 500nm. ThT fluorescence indicative of total fibril content (C), 5μg of extract was analyzed. Statistical significance was determined by unpaired Student’s t-test ****p<0.0001.

Figure 2

Quantification of amyloidoma-associated Dylight800 fluorescence intensity as normalized fluorescence. Data are representative of 2 experiments with n=8-12 animals per group. Mean and standard deviation at each time point shown. Data were analyzed using Student’s t-test at each time point to determine significance **, p<0.01; ****, p<0.001.

Figure 3

Immunophenotypic evaluation of immune infiltrate into ALλ(CLA) or ALκ(TAL) amyloidomas. Flow cytometry was used to identify single cell suspensions isolated from indicated tissues Day 1 (A) and Day 8 (B) post amyloid implantation. Cell surface markers used to identify each indicated population are detailed on the y-axis of each panel. The gating strategy used for analysis can be found in Supplementary Figure 1 . Excised amyloidoma tissues sections were stained with H&E to identify immune cell infiltrates (C, D). Images are representative of 3-4 mice per group. Black scale bars represent 50μm. Data represent the mean ± standard deviation from one experiment which is representative of two experimental replicates, with n=5-6 animals per group per experiment. Statistical significance was determined by Student’s t test, ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. ns, not significant.

Figure 4

Spatial analysis of intra-amyloid neutrophil recruitment. Immunohistochemistry was used to specifically analyze neutrophils in tissues 8 days post the implantation of either ALλ(CLA) (A) or ALκ(TAL) (B) amyloidomas. H&E staining was performed to identify polymorphonuclear cells and other immune infiltrate. Ly6G staining was used to identify intact neutrophils and neutrophil membrane remnants. Citrullinated histone 3 staining was used to identify neutrophil extracellular traps. Data is representative of two experimental replicates with 5 animals per group. Scale bars = 50μm.

Figure 5

Amyloidoma cytokine expression in ALλ(CLA) or ALκ(TAL) amyloidomas. NU/NU mice were treated as in Figure 3 and implanted with ALκ(TAL) or ALλ(CLA) amyloidomas. 8 days following amyloid implantation amyloidomas were harvested and cytokine expression determined using a BioLegend, LEGENDplex™ panel. n=1 with 3 mice per group. Statistical significance was determined by unpaired Student’s t-test *p<0.05, **p<0.01. ns, not significant.

Figure 6

Evaluation of the neutrophil-amyloid relationship. Flow cytometry analysis of human neutrophil (A) or thioglycolate-elicited murine neutrophil (B) phagocytosis of pHrodoRed-labeled ALλ(CLA) extract or ALκ(TAL) extract. Primary neutrophil ROS productions was analyzed by flow cytometry in the presence of indicated amyloid material. (C). Effect of H₂O₂ treatment of amyloid extract and fibril degradation (D). Statistical significance was determined by unpaired Student’s t-test ****p<0.0001.

Figure 7

Neutrophil depletion impedes ALκ(TAL) resolution in vivo. Immunodeficient mice were implanted with Dylight800 labeled ALκ(TAL) amyloidomas and fluorescence measured over time (A, B, D, E),. 18 days post-implantation, amyloidomas were extracted, and amyloid area was determined by ImageJ (C). Mice received either the anti-Ly6G (1A8) neutrophil depletion antibody or vehicle control treatment. Data is representative of 2 experiments with 8-12 mice per group. Statistical significance was determined by unpaired Student’s t-test *p<0.05, **p<0.01, ***p<0.001.

Figure 8

Amyloidoma cytokine expression in neutrophil depleted or non-depleted mice. NU/NU mice were treated as in Figure 5A and implanted with ALκ(TAL) amyloidomas. 1 (A) and 4 (B) days following amyloid implantation amyloidomas were harvested and cytokine expression determined using a BioLegend, LEGENDplex™ panel. n=1 with 3-4 mice per group. Statistical significance was determined by unpaired Student’s t-test *p<0.05, **p<0.01, ***p<0.001.

Figure 9

Neutrophil depletion delays amyloidoma microenvironment maturation. Excised amyloidoma tissues sections were stained with H&E to identify immune cell infiltrates. Images representative of n=4 mice per group. Black scale bars represent 50μm.