Elimination of activating Fcγ receptors in spontaneous autoimmune peripheral polyneuropathy model protects from neuropathic disease

Abstract

Spontaneous autoimmune peripheral polyneuropathy (SAPP) is a reproducible mouse model of chronic inflammatory peripheral neuropathy in female non-obese diabetic mice deficient in co-stimulatory molecule, B7-2 (also known as CD86). There is evidence that SAPP is an interferon-γ, CD4+ T-cell-mediated disorder, with autoreactive T-cells and autoantibodies directed against myelin protein zero involved in its immunopathogenesis. Precise mechanisms leading to peripheral nerve system inflammation and nerve injury including demyelination in this model are not well defined. We examined the role of activating Fc-gamma receptors (FcγRs) by genetically ablating Fcγ-common chain (Fcer1g) shared by all activating FcγRs in the pathogenesis of this model. We have generated B7-2/ Fcer1g-double null animals for these studies and found that the neuropathic disease is substantially ameliorated in these animals as assessed by behavior, electrophysiology, immunocytochemistry, and morphometry. Our current studies focused on characterizing systemic and endoneurial inflammation in B7-2-null and B7-2/ Fcer1g-double nulls. We found that accumulation of endoneurial inflammatory cells was significantly attenuated in B7-2/ Fcer1g-double nulls compared to B7-2-single nulls. Whereas, systemically the frequency of CD4+ regulatory T cells and expression of immunosuppressive cytokine, IL-10, were significantly enhanced in B7-2/ Fcer1g-double nulls. Overall, these findings suggest that elimination of activating FcγRs modulate nerve injury by altering endoneurial and systemic inflammation. These observations raise the possibility of targeting activating FcγRs as a treatment strategy in acquired inflammatory demyelinating neuropathies.

Fig 1. Deficiency of activating FcγRs ameliorates immune mediated neuropathy in female B7-2 null NOD mice.

A &B. Light micrographs of toluidine blue-stained transverse sciatic nerve sections showing signs of demyelination (onion bulbs, arrow; demyelinated axons), axonal degeneration (decrease in myelinated nerve fibers), and macrophage infiltration (arrow head) in B7-2 null NOD mice (A); in contrast, minimal nerve injury and inflammation were seen in Fcer1g^-/- B7-2^-/- NOD mice (B). C & D. Morphometry on epon sections of sciatic nerves showing there are significantly more myelinated nerve fibers (MF) in Fcer1g^+/- B7-2^-/- and Fcer1g^-/- B7-2^-/- mice, compared to B7-2 single null NOD mice (Fcer1g^+/+ B7-2^-/-;) at age of 35 weeks (C; n = 25 for each genotype) and 55 weeks (D; n = 20 for each genotype). E & F. There are significantly reduced numbers of demyelinated fibers (DMF) in Fcgr^+/-/B7-2^-/- and Fcgr^-/-/B7-2^-/- compared to B7-2 null NOD mice at age of 35 weeks (E; n = 25 for each genotype) and 55 weeks (F; n = 20 for each genotype). Scale bar = 10μm. *P < 0.05.

Fig 2. Clinical assessment shows improvement in motor nerve function after deletion of activating FcγRs in female SAPP mice.

A & B. Behavior (Rotarod; A) and electrophysiology (B) tests showing protection from disease in double knockouts (Fcer1g^-/- B7-2^-/-), intermediate neuropathic phenotype in Fcer1g^+/- B7-2^-/-, and full-blown neuropathy in Fcer1g^+/+ B7-2^-/- mice. Data acquired from age 20–55 weeks old female mice. *P < 0.05 (n = 20).

Fig 3. Effects of activating FcγRs depletion on endoneurial and systemic inflammation in female B7-2 null NOD mice.

A. Immunocytochemistry for macrophage marker CD68 and lymphocyte marker CD3 showing robust inflammation in Fcer1g^+/+ B7-2^-/- and minimal inflammation in Fcer1g^-/- B7-2^-/-sciatic nerves. B & C. Quantification analysis of macrophage counts (B) (n = 20) and lymphocyte counts (C) (n = 20); D. Flow cytometry analysis showing percentage of NK cells (CD45+/CD335+) in spleen samples of Fcer1g^-/- B7-2^-/- mice are significantly lower than in those of Fcer1g^+/+ B7-2^-/- mice (n = 5). E. The depletion of activating FcγRs gene results in an higher percentage of regulatory T cells (CD4+/FoxP3+) and IL-10-secreting T cells (CD4+/IL-10+) in spleen samples of Fcer1g^-/- B7-2^-/- NOD mice (n = 5). According to their forward and side scatter properties, lymphocytes were selected from the total splenocytes, dead cells and cellular debris were excluded. It was then followed by gating on CD4, and subsequent analysis for FoxPx3 or IL-10 expression. Scale bar = 10μm. *P < 0.05.

Fig 4. Activating FcγRs depletion suppresses P0 specific T cell response and LPS-induced macrophage activation.

A. Images from IFN-γ ELISPOT assay showing positive spots from Fcer1g^+/+ B7-2^-/- and Fcer1g^-/- B7-2^-/- splenocytes in the presence of P0 peptide. B. Quantitative ELISPOT assay analysis showing that IFN-γ positive P0 peptide reactive cells are significantly reduced in Fcer1g^-/- B7-2^-/- compared to Fcer1g^+/+ B7-2^-/- mice (n = 7 for each genotype). C. Representative flow plots showing the differences in proportion of TNFa or IL-6 producing CD11b+ cells from Fcer1g^+/+ B7-2^-/- and Fcer1g^-/- B7-2^-/- mice with or without LPS (Cont) stimulation. D. Quantification of flow cytometry analysis showing the expression of proinflammatory cytokines in CD11b+ cells from Fcer1g^+/+ B7-2^-/- and Fcer1g^-/- B7-2^-/- mice with or without LPS (Cont) stimulation (n = 8). *P < 0.05.

Fig 5. Schematic diagram of the postulate that deletion of activating FcγRs alters macrophage activation and effector functions.

Conceptual diagram showing our hypothesis that deletion of Fcγ-common chain expression and associated activating FcγRs on macrophage lineage cells alters their pro-inflammatory state and effector functions including antigen presentation to T cells, which in turn reduces the endoneurial inflammatory burden and nerve injury in SAPP model.