Figure 4. EAO of Treg-depleted DEREG mice is characterized by M1-like macrophage infiltration, immune complex deposition, and disrupted Sertoli cell barrier.

(A) Testis weight at 8 weeks after DT treatment. (B) Increased interstitial F4/80⁺ macrophages (red) and reduced seminiferous tubule diameter and ZAN⁺ spermatids (green) in DEREG mice with EAO compared with control mice. Original magnification, ×100. (C) Most macrophages of control mice have the characteristic phenotype of M2 macrophages, including the expression of IL-4Rα, CD206, and F4/80 (left), whereas macrophages of Treg-depleted DEREG mice have an M1-like phenotype of high iNOS and MHC class II expression (right); data are expressed as a ratio of absolute numbers (right), where each symbol represents cells from 2 testes of 1 mouse. (D) Mouse IgG immune complexes (green) in the DEREG mouse testes with EAO are located both outside and inside seminiferous tubules relative to the occludin⁺ Sertoli cell barrier (red). Original magnification, ×300. (E) Biotin (red) exclusion assay in testes of control mice (top) and Treg-depleted DEREG mice (bottom). Original magnification, ×100. (F) Western blot analysis of extractable tight junction proteins of the Sertoli cell barrier from testes of control and Treg-depleted DEREG mice. This is a composite data set of 8 parallel blots wherein 4 control and Treg-depleted DEREG mouse testis samples were analyzed by using corresponding specific Abs in a single experimental session to avoid interexperimental variations. (G) IF staining of murine IgG (green) and rabbit IgG (red) in the testis of a DEREG mouse with EAO at 19 hours after i.p. injection of rabbit Abs to LDH3. Note rabbit (red) and mouse (green) immune complexes were admixed or colocalized inside the seminiferous tubule (arrow). Dotted line, seminiferous tubule boundary. Original magnification, ×800. Data are from 2–12 independent experiments. *P < 0.05; ***P < 0.001, Mann-Whitney U test (A and C).