The clinical-radiological paradox in multiple sclerosis: myth or truth?

Background Multiple sclerosis (MS) is an inflammatory, degenerative, demyelinating disease that ranges from benign to rapidly progressive forms. A striking characteristic of the disease is the clinical-radiological paradox. Objectives The present study was conducted to determine whether, in our cohort, the clinical-radiological paradox exists and whether lesion location is related to clinical disability in patients with MS. Methods Retrospective data from 95 patients with MS (60 women and 35 men) treated at a single center were examined. One head-and-spine magnetic resonance imaging (MRI) examination from each patient was selected randomly, and two independent observers calculated lesion loads (LLs) on T2/fluid attenuation inversion recovery sequences manually, considering the whole brain and four separate regions (periventricular, juxtacortical, posterior fossa, and spinal cord). The LLs were compared with the degree of disability, measured by the Kurtzke Expanded Disability Status Scale (EDSS), at the time of MRI examination in the whole cohort and in patients with relapsing-remitting (RR), primarily progressive, and secondarily progressive MS. Results High LLs correlated with high EDSS scores in the whole cohort (r = 0.34; p < 0.01) and in the RRMS group (r = 0.27; p = 0.02). The EDSS score correlated with high regional LLs in the posterior fossa (r = 0.31; p = 0.002) and spinal cord (r = 0.35; p = 0.001). Conclusions Our results indicate that the clinical-radiological paradox is a myth and support the logical connection between lesion location and neurological repercussion

P2X7 receptor drives Th1 cell differentiation and controls the follicular helper T cell population to protect against <i>Plasmodium chabaudi</i> malaria

<div><p>A complete understanding of the mechanisms underlying the acquisition of protective immunity is crucial to improve vaccine strategies to eradicate malaria. However, it is still unclear whether recognition of damage signals influences the immune response to <i>Plasmodium</i> infection. Adenosine triphosphate (ATP) accumulates in infected erythrocytes and is released into the extracellular milieu through ion channels in the erythrocyte membrane or upon erythrocyte rupture. The P2X7 receptor senses extracellular ATP and induces CD4 T cell activation and death. Here we show that P2X7 receptor promotes T helper 1 (Th1) cell differentiation to the detriment of follicular T helper (Tfh) cells during blood-stage <i>Plasmodium chabaudi</i> malaria. The P2X7 receptor was activated in CD4 T cells following the rupture of infected erythrocytes and these cells became highly responsive to ATP during acute infection. Moreover, mice lacking the P2X7 receptor had increased susceptibility to infection, which correlated with impaired Th1 cell differentiation. Accordingly, IL-2 and IFNγ secretion, as well as T-bet expression, critically depended on P2X7 signaling in CD4 T cells. Additionally, P2X7 receptor controlled the splenic Tfh cell population in infected mice by promoting apoptotic-like cell death. Finally, the P2X7 receptor was required to generate a balanced Th1/Tfh cell population with an improved ability to transfer parasite protection to CD4-deficient mice. This study provides a new insight into malaria immunology by showing the importance of P2X7 receptor in controlling the fine-tuning between Th1 and Tfh cell differentiation during <i>P</i>. <i>chabaudi</i> infection and thus in disease outcome.</p></div

Splenic B6 or <i>P2rx7</i>^-/- CD4 cell co-transfer and protection against <i>Pc</i> infection.

<p>(A-D) Naïve CD4⁺ cells from B6 (CD45.1) and <i>P2rx7</i>^-/- (CD45.2) female mice were co-transferred into <i>Cd4</i>^-/- female mice that were then infected with 1 × 10⁶ <i>Pc</i>-iRBCs. Splenic CD4⁺ cells were analyzed at 30 days p.i. Non-infected mice were used as controls (day 0). The data were expressed as means ± SD (<i>n</i> = 5) of one representative experiment out of three. Significant differences were for the (*) indicated groups with <i>p</i> < 0.05, using the Mann Whitney U test (NS, not significant). (A) A schematic illustration of the experimental protocol is shown. (B) Dot plot shows CD45.1⁺CD4⁺ and CD45.1^-CD4⁺ cells at 30 days p.i. CD45.1⁺CD4⁺ and CD45.1^-CD4⁺ cell numbers per spleen are shown in the column bar graph. (C) Contour plots show PD1 and CD39 expression in CD4⁺ cells. PD1^hiCD39^loCD4⁺ and PD1^loCD39^hiCD4⁺ cell percentages are shown in the column bar graphs. (D) Histograms show T-bet and Bcl6 expression in PD1^hiCD39^loCD45.1⁺CD4⁺, PD1^loCD39^hiCD45.1⁺CD4⁺, PD1^hiCD39^loCD45.1^-CD4⁺ and PD1^loCD39^hiCD45.1^-CD4⁺cells. FMO controls are shown in the histograms. The MFIs of T-bet and Bcl6 expression are shown in the column bar graphs. (E-F) CD4⁺ cells from B6 and <i>P2rx7</i>^-/- female mice at 20 days p.i. were transferred into <i>Cd4</i>^-/- female mice that were infected with 1 × 10⁵ <i>Pc</i>-iRBCs. <i>Cd4</i>^-/- mice transferred with naïve B6 cells were used as controls. The data were expressed as means ± SD (<i>n</i> = 4–6) of one representative experiment out of three. Significant differences were observed for the (*) mice transferred with B6 cells at 0 and 20 days p.i. and (**) mice transferred with B6 and <i>P2rx7</i>^-/- cells at 20 days p.i. with <i>p</i> < 0.05, using the Mann Whitney U test. (E) A schematic illustration of the experimental protocol is shown. (F) Parasitemia curves are shown.</p

Parasitemia and clinical parameters in B6 and <i>P2rx7</i>^-/- mice infected with <i>Pc</i> and P. <i>yoelii</i> 17NL parasites.

<p>(A-C) B6 and <i>P2rx7</i>^-/- mice were infected with 1 × 10⁶ <i>Pc</i>-iRBCs. The data were expressed as means ± SD (<i>n</i> = 4–5) of one representative experiment out of three. Significant differences were observed for the (*) B6 and <i>P2rx7</i>^-/- groups with <i>p</i> < 0.05, using (A and C) the Mann Whitney U test or (B) the long-rank test. (A) Parasitemia curves are shown. (B) Survival curves are shown. (C) Hemoglobin serum (Hb) concentration, body weight and body temperature were monitored daily. Variation in body weight relative to day 0 is shown. (D) B6 and <i>P2rx7</i>^-/- mice were infected with 1 × 10⁵ <i>P</i>. <i>yoelii</i> 17XNL-iRBCs. The data were expressed as means ± SD (<i>n</i> = 4–5) of one representative experiment out of three. Significant differences were observed for the (*) B6 and <i>P2rx7</i>^-/- groups with <i>p</i> < 0.05, using the Mann Whitney U test. Parasitemia curves are shown.</p

Splenic CD4 T cell responses in acutely infected B6 and <i>P2rx7</i>^-/- mice.

<p>(A-G) B6 and <i>P2rx7</i>^-/- mice were analyzed at 4, 5 and 7 days p.i. with 1 × 10⁶ <i>Pc</i>-iRBCs. Naïve mice were used as controls (day 0). Female mice were used in the experiments with the exception that females and males were compared. The data were expressed as means ± SD (<i>n</i> = 3–4) of one representative experiment out of three. Significant differences were observed for the (*) indicated groups with <i>p</i> < 0.05, using the Mann Whitney U test (NS, not significant). (A) EB-stained CD4⁺ blood cell percentages were determined by flow cytometry, before and after iRBC rupture. The blood samples were collected at 9 a.m. (1.3 ± 0.3% iRBCs at 4 days p.i. and 7.6 ± 0.3% iRBCs at 5 days p.i.; >95% trophozoites and schizonts) and 2 p.m. (7.3 ± 1.2% iRBCs at 4 days p.i. and 15.0 ± 3.7% iRBCs, at 5 days p.i.; >95% ring forms). (B) Splenocytes were stimulated or not with 25–500 μM ATP. EB-stained CD4⁺ cell percentages were determined by flow cytometry. Horizontal gray bar represents the confidence interval obtained with <i>P2rx7</i>^-/- cells. (C) CFSE-stained CD4⁺ cells and APCs (splenocytes from naïve nude mice), at a 1:1 ratio, were stimulated or not with iRBCs (1 splenocyte/ 4 iRBCs). Histograms show CFSE-stained CD4⁺ cells. CFSE^loCD4⁺ cell percentages are shown in the column bar graph. (D) Splenocytes were stimulated or not with iRBCs (1 splenocyte/ 3 iRBCs). Contour plots show intracellular IFNγ- and IL-10-stained CD4⁺ cells. IFNγ⁺IL-10^- and IFNγ⁺IL-10⁺ cell percentages in CD4⁺ cells are shown in the column bar graphs. (E) CD4⁺ cell numbers per spleen were determined by flow cytometry. (F) IFNγ⁺CD4⁺ and IL-10⁺CD4⁺ cell numbers per spleen were determined by flow cytometry. (G) IgM-, IgG2c- and IgG1-secreting cell numbers per spleen were determined by ELISPOT assay.</p

Splenic Tfh cell responses in infected B6 and <i>P2rx7</i>^-/- mice.

<p>(A-G) B6 and <i>P2rx7</i>^-/- female mice were analyzed at 7,14, 20, 30, 50, 100 and 130 days p.i. with 1 × 10⁶ <i>Pc</i>-iRBCs. Naïve mice were used as controls (day 0). The data were expressed as means ± SD (<i>n</i> = 3–5) of one representative experiment out of three. Significant differences were observed for the (*) B6 and <i>P2rx7</i>^-/- groups with <i>p</i> < 0.05, using the Mann Whitney U test (NS, not significant). (A) Hematoxilyn-eosin stained sections show splenic follicular hyperplasia in <i>P2rx7</i>^-/- mice (40x magnification; bar scales correspond to 200 μm). (^#) The mean areas of lymphoid follicles are shown. (B) Confocal immunofluorescence images (100x magnification; bar scales correspond to 400 μm) of splenic sections are shown. Tissue slices were stained for CD19 (green), CD4 (red) and GL7 (blue). (C) CD19⁺ and Fas⁺GL7⁺CD19⁺ cell numbers per spleen were determined by flow cytometry. (D) Contour plots show PD1, ICOS and Bcl6 <i>versus</i> CXCR5 expression in CD4⁺ cells. PD1⁺CXCR5⁺CD4⁺, ICOS⁺CXCR5⁺CD4⁺ and Bcl6⁺CXCR5⁺CD4⁺ cell numbers per spleen are shown in the column bar graphs. (E) PD1⁺CXCR5⁺CD4⁺ cell numbers per spleen were determined by flow cytometry. (F) IL-21 concentrations were determined by ELISA in the supernatants of splenocytes stimulated or not with iRBCs (splenocyte/3 iRBCs). (G) Anti-parasite IgM and IgG2c serum concentrations were determined by ELISA.</p

Splenic Th1 cell responses in chronically infected B6 and <i>P2rx7</i>^-/- mice.

<p>(A-F) B6 and <i>P2rx7</i>^-/- female mice were analyzed at 20, 30 and 50 days p.i. with 1 × 10⁶ <i>Pc</i>-iRBCs. Naïve mice were used as controls (day 0). The data were expressed as means ± SD (<i>n</i> = 3–5) of one representative experiment out of three. Significant differences were observed for the (*) indicated groups with <i>p</i> < 0.05, using the Mann Whitney U test (NS, not significant). (A) Splenocytes were stimulated or not with 25–500 μM ATP. EB-stained CD4⁺ cell percentages were determined by flow cytometry. Horizontal gray bar represents the confidence interval obtained with <i>P2rx7</i>^-/- cells. (B) CD4⁺ cell numbers per spleen were determined by flow cytometry. (C) Splenocytes were stimulated or not with iRBCs (1 splenocyte/ 3 iRBCs). IFNγ⁺IL-10^-, IFNγ⁺IL-10⁺ and IFNγ^-IL-10⁺ cell percentages in CD4⁺ cells were determined by flow cytometry. (D) IFNγ and IL-10 concentrations were determined by ELISA in the supernatants of splenocytes described in E, which were stimulated or not with 100–300 μM ATP. (E) Histograms show T-bet expression in CD4⁺ cells. FMO controls are shown in the histograms. The MFIs of T-bet expression are shown in the scatter plots. (F) Contour plots show T-bet and P2X7 expression in CD4⁺ cells. T-bet⁺P2X7⁺ cell percentages in CD4⁺ cells are shown in the column bar graph.</p

Splenic Th1 and Tfh cell differentiation in <i>Cd4</i>^-/- mice adoptively transferred with B6 or <i>P2rx7</i>^-/- CD4 cells and infected with <i>Pc</i> parasites.

<p>(A-G) Naïve CD4⁺ cells from B6 and <i>P2rx7</i>^-/- female mice were transferred into <i>Cd4</i>^-/- female mice that were infected with 1 × 10⁶ <i>Pc</i>-iRBCs 7 days later. Splenic CD4⁺ cells were analyzed at 7 and 30 days p.i. Non-infected mice were used as controls (day 0). The data were expressed as means ± SD (<i>n</i> = 3–5) of one representative experiment out of three. Significant differences were observed for the (*) B6 and <i>P2rx7</i>^-/- groups with <i>p</i> < 0.05, using the Mann Whitney U test (NS, not significant). (A) A schematic illustration of the experimental protocol is shown. (B) Parasitemia curves are shown. Arrows indicate the days of chloroquine treatment. (C) Immunofluorescence images (100x magnification bar scales correspond to 100 μm) showing cytosolic calcium levels in purified CD4⁺ cells were obtained using the calcium flux assay. (^#) The mean corrected total cell fluorescence (CTCF) of CD4⁺ cells are shown. (D) Dot plots show IL-2-secreting CD4⁺ cells. IL-2-secreting CD4⁺ cell numbers per spleen are shown in the column bar graph. (E) Contour plots show intracellular IFNγ-stained CD4⁺ cells. IFNγ⁺CD4⁺ cell numbers per spleen are shown in the column bar graph. (F) Histograms show T-bet expression in CD4⁺ cells. FMO controls are shown in the histograms. The MFIs of T-bet expression are shown in the column bar graphs. (G) Contour plots show PD1 and Bcl6 <i>versus</i> CXCR5 expression in CD4⁺ cells. PD1⁺CXCR5⁺CD4⁺ and Bcl6⁺CXCR5⁺ cell percentages in CD4⁺ cells are shown in the column bar graphs.</p