Additional file 1: of Paracoccidioides brasiliensis infection promotes thymic disarrangement and premature egress of mature lymphocytes expressing prohibitive TCRs

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Increased protein and mRNA expression of MHC-I and PirB in the spinal cord following PNS damage.

<p>Mice were submitted to sciatic nerve transection. One week after lesion (wal), the spinal cord on the ipsilateral side was analyzed for MHC-I and PirB protein and gene expression. Uninjured mice were used as a control. MHC-I (A) and PirB (B) protein quantification by the integrated density of pixels method, applying the ratio lesioned/unlesioned. mRNA expression of the MHC-I β2 m chain (C) and PirB (D), relative to the control group. Data are presented as the mean ± SEM. n = 6 in each time point. *p<0.05; **p<0.01 according to the unpaired <i>t</i> test. E) Control neuron displaying cytoplasmic vesicles containing MHC-I molecules. Scale bar: 20 μm.</p

PirB expression in the sciatic nerve.

<p>Representative images of sciatic nerves double immunolabeled for PirB and A-D) Iba1 (macrophage marker), E) neurofilament (NF, axonal marker), or F and G) S100 (Schwann cell marker). A) PirB expression by macrophages was mostly restricted to round-shaped cells. Inset: macrophage expressing PirB (arrowhead); DAPI stained nuclei. B) Orthogonal projection of PirB⁺ macrophages. C) Macrophages from the undamaged nerve did not express PirB, whereas D) macrophages from the adjacent adipose tissue were PirB⁺ (arrowheads). Asterisks indicate adipocytes; DAPI stained nuclei, PC: phase contrast. E) Low or absent PirB expression by axons. In the detail an axon wrapped by PirB labeled Schwann cells (arrowheads). F) Schwann cells strongly expressing PirB (arrowheads in the detail). G) Transverse section of nerve fibers (arrowheads), showing PirB immunolabeling associated with the myelin sheath. Asterisk indicates the space occupied by the axon. Time-points: A and B, 2 weeks after lesion (wal); C and D, zero wal; E and G, 4 wal; F, 8 wal. Scale bars: A, 100 μm; B, 20 μm; C-F, 50 μm; G, 10 μm. Insets: A and F, 20 μm; B, 10 μm.</p

Crushed sciatic nerve CD8 T cell immunolabeling, quantification and phenotyping.

<p>A, B) Crushed sciatic nerve (2 weeks after lesion) was immunolabeled for CD8 T cells (green) and neurofilaments (axonal marker, red). Note CD8 T cells in the endoneural environment (B, detail). Scale bars: A, 50 μm; B, 20 μm. C-E) Crushed sciatic nerves were dissected at 2, 4 and 8 weeks after lesion (wal) and submitted to flow cytometry procedures. C) Representative graphs of CD8 T cell quantification (D) and cytokine expression (E). Note that the frequency of CD8 T cells diminishes over time (D), and these lymphocytes gradually express less INF-γ and more IL-10 (E). Data are presented as the mean ± SEM. n = 6 in each time point. **p<0.01; ***p<0.001 according to the one-way ANOVA followed by Bonferroni post-tests. F-H) H&E stained nerves at 2 (F), 4 (G) and 8 (H) weeks after lesion (wal), evidencing inflammation resolution. Scale bars: 50 μm.</p

MHC-I and PirB Upregulation in the Central and Peripheral Nervous System following Sciatic Nerve Injury

<div><p>Major histocompatibility complex class one (MHC-I) antigen-presenting molecules participate in central nervous system (CNS) synaptic plasticity, as does the paired immunoglobulin-like receptor B (PirB), an MHC-I ligand that can inhibit immune-cells and bind to myelin axon growth inhibitors. Based on the dual roles of both molecules in the immune and nervous systems, we evaluated their expression in the central and peripheral nervous system (PNS) following sciatic nerve injury in mice. Increased PirB and MHC-I protein and gene expression is present in the spinal cord one week after nerve transection, PirB being mostly expressed in the neuropile region. In the crushed nerve, MHC-I protein levels increased 2 weeks after lesion (wal) and progressively decreased over the next eight weeks. The same kinetics were observed for infiltrating cytotoxic T lymphocytes (CTLs) but not for PirB expression, which continuously increased. Both MHC-I and PirB were found in macrophages and Schwann cells but rarely in axons. Interestingly, at 8 wal, PirB was mainly restricted to the myelin sheath. Our findings reinforce the participation of MHC-I and PirB in CNS plasticity events. In contrast, opposing expression levels of these molecules were found in the PNS, so that MHC-I and PirB seem to be mostly implicated in antigen presentation to CTLs and axon myelination, respectively.</p></div

Increased expression of MHC-I molecules in the sciatic nerve after crushing.

<p>Mice were submitted to the crushing of the sciatic nerve. 2, 4, and 8 weeks after lesion (wal), nerves were dissected out and analyzed by immunofluorescence for MHC-I expression. Uninjured mice were used as a control (zero wal). A) representative images of MHC-I labeling at all time-points. Scale bar: 100 μm. B) MHC-I quantification by the integrated density of pixels method (lesioned/unlesioned). Data are presented as the mean ± SEM. n = 6 in each time point. ***p<0.001 according to the one-way ANOVA, followed by Bonferroni post-tests.</p

Sciatic nerve MHC-I expression by distinct cellular sources after crushing.

<p>Double immunolabeling of MHC-I molecules and A) neurofilament (NF, axonal marker), or B) S100 protein (Schwann cell marker) or C) Iba1 protein (macrophage marker) after crushing. A) Most of the axons expressed zero or low MHC-I immunolabeling. Inset: MHC-I⁺ axon (arrowheads). B) MHC-I expression by Schwann cells. Inset: MHC-I labeled Schwann cell (arrowheads). C) Macrophages expressing MHC-I after injury. In detail, MHC-I labeled macrophages (arrowheads), one of which with cytoplasmic MHC-I immunolabeling. D) MHC-I expression by endothelial cells. A blood vessel is highlighted (dashed lines). Nuclei are DAPI positive, NF: neurofilament, CD31: endothelial cell adhesion molecule. Time-points: A and D, 4 weeks after lesion (wal); B and C, 2 wal. Scale bars: A and D, 50 μm; B and C, 100 μm; insets, 20 μm.</p

Spinal cord PirB immunolabeling is stronger in neuronal soma and processes.

<p>Following sciatic nerve transection, lumbar spinal cord sections were double-immunolabeled for PirB and A) Iba1 (microglia marker), B) GFAP (astrocyte marker), or C) NeuN (neuronal soma marker). Non-operated mice were used as a control. A) Low or null expression of PirB by microglia, either before or after nerve axotomy. B) Absent or low punctal expression of PirB by gray matter astrocytes. PirB immunolabeling could be visualized in white matter astrocytes (inset, arrowheads). C) PirB expression by neuronal soma surface and processes, under basal conditions and after injury. Arrowheads in A, B and C indicate neuronal soma expressing PirB. Scale bars: A-C, 50 μm; D, 20 μm.</p

PirB expression in the crushed sciatic nerve.

<p>A) Representative images of PirB immunostained sciatic nerve at 2, 4, and 8 weeks after lesion (wal). Undamaged nerve was used as a control (zero wal). Scale bar: 100 μm. B) PirB expression quantification by the integrated density of pixels method, where the ratio lesioned/unlesioned was employed. Data are presented as the mean ± SEM. n = 6 in each time point. *p<0.05; **p<0.01; ***p<0.001 according to the one-way ANOVA, followed by Bonferroni post-tests.</p

Prolonged <i>Paracoccidioides brasiliensis</i> infection leads to thymic atrophy and intense fungal burden.

<p>Male BALB/c mice (n = 5 mice/group for each analyses per experiment replicate) were inoculated with 5x10⁶ Pb18 yeasts contained in PBS or with only PBS (control group), intraperitoneally. One hundred and twenty days after inoculation, mice were killed and the thymus removed for analysis. A) Thymus from the 120dpi group was smaller in size and weighted less than the naive group. (B) Hematoxilin-Eosin staining showed histologic disorganization in 120dpi, and no evidence of typical cortical (C) and medullary (M) regions, while naive mice showed normal histologic distribution. In more detail below, giant cells and granuloma formation is present in 120dpi. Silver impregnation revealed massive fungal infiltration in the thymic medulla in 120dpi. Statistical analysis was carried out with Student’s t-test. **p<0.01. Results are expressed by Mean ± SEM. Images are representative of three independent experiments with similar results. The images were taken in an Olympus BX50. Magnification 40x (upper and lower panels); 100x (middle panel).</p