Fig 1.
RSA59 infection alters the expression of CD40L in WT and CD4-/- mouse brains.
(A) Schematic diagram representing the kinetics of MHV-A59/RSA59 viral RNA, infectious particles, and phases of immune response correlating with acute phase neuroinflammation, the bridging of innate and adaptive immune responses, and chronic phase peak of demyelination following intracranial inoculation. Scale-arbitrary. (B) CD40L transcript abundance was determined in whole brain lysates from RSA59 infected (25000 PFUs) WT mice on days 5 and 10 p.i. by qRT-PCR. Results were normalized to β-Actin, compared with mock-infected control, and expressed as mean ± SEM. (C, D) CD40L protein expression was determined by immunoblot analysis in whole brain lysates of RSA59 infected WT mice on days 5 and 10 p.i. Results were normalized to γ-Actin, compared with mock-infected control, and expressed as mean ± SEM. (E) Whole brain lysates of RSA59 infected WT and CD4-/- mice were subjected to qRT-PCR, and CD40L transcript abundance was compared between the two mouse strains on days 5 and 10 p.i. *Asterisk represents statistical significance calculated using unpaired Student’s t-test, p<0.05 was considered significant. **p<0.01, ****p<0.0001. Data is represented from 3 independent biological experiments, N = 3.
Fig 2.
CD40L deficiency makes mice more susceptible to RSA59 infection.
WT (N = 17) and CD40L-/- mice (N = 21) were infected with RSA59 (25000 PFUs) and monitored daily for (A) survival, (B) weight change, (C) development of the clinical disease, and (D) viral titers in brains. Clinical scores were assigned by a relative scale of 0–4 as described in Materials and Methods. Results were expressed as mean ± SEM. *Asterisk represents statistical significance calculated using unpaired Student’s t-test, p<0.05 was considered as significant. Statistical significance for the survival curve was determined by Log-rank (Mantel-Cox) test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 3.
CD40L deficiency results in reduced Iba1+ microglial inflammation in the brain.
On day 5 p.i., sections of brains (A) from RSA59 infected (25000 PFUs) WT and CD40L-/- mice were stained with H&E and immunohistochemically for Iba1. The boxed areas are shown at higher magnification below the corresponding brain midsagittal sections. Black arrows in the zoomed sections mark characteristic perivascular cuffing, and black arrowheads mark microglial nodule formation. High magnification images corresponding to blue-lined rectangles from the cortical regions show the distinct morphology and distribution of Iba1+ cells. Scale bar, 500μm, 50μm. (B) Shows quantification of Iba1 staining in the brain. Results were expressed as mean ± SEM from 3–4 independent biological experiments (N = 4–5). *Asterisk represents statistical significance calculated using unpaired Student’s t-test and Welch correction, p<0.05 was considered significant. ****p<0.0001.
Fig 4.
CD40L deficiency causes impaired accumulation and activation of peripheral monocyte/macrophage and the brain resident microglia at the acute phase of inflammation.
On day 5 p.i., brains from MI and RSA59 infected (25000 PFUs) WT and CD40L-/- mice were harvested for flow cytometry analysis and stained for CD45, CD11b, MHCII, CX3CR1, and CD40. The green color denotes WT, and blue indicates CD40L-/- mice. (A) Representative flow cytometry contour plots showing percentages of overall CD45hi and CD45lo cell population after gating on singlets followed by live cells and absolute cell numbers from infected sets are represented in (B) comparing WT and CD40L-/- from MI and infected mice groups. Percentages of CD45 gated cells assessed for CD45hiCD11b+ (peripheral derived monocyte/macrophage) and CD45loCD11b+ (brain resident microglia) from infected sets are presented in flow cytometry plots (C), and absolute numbers of MI and infected sets are graphically represented in (D) comparing WT and CD40L-/- groups. Representative flow cytometry contour plots indicate percentages of MHCII+ (E), CX3CR1(G), and CD40 (I) expressing CD45hiCD11b+ and CD45loCD11b+ cells comparing infected WT and CD40L-/- mice, and the absolute numbers of MI and infected sets are graphically represented in F, H, and J, respectively. Results were expressed as mean ± SEM from 3 independent biological experiments (N = 4). *Asterisk (MI WT v/s infected WT) and #hash (infected WT v/s infected CD40L-/-) represents statistical significance calculated using unpaired Student’s t-test and Welch correction, p<0.05 was considered significant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 5.
CD40L deficiency results in reduced activation of monocytes/macrophages and microglia on day 10 p.i.
On day 10 p.i., cell suspensions from brains of MI and RSA59 infected (10000 PFUs) WT and CD40L-/- mice were analyzed as described in Fig 4 by flow cytometry following staining for CD45, CD11b, MHCII, CX3CR1, and CD40. The green color denotes WT, and blue indicates CD40L-/- mice. (A) Shows percentages of CD45hiCD11b+ (peripheral derived monocyte/macrophage) and CD45loCD11b+ (brain resident microglia) from infected sets in representative contour plots. (B) shows quantification of absolute numbers comparing MI and infected WT and CD40L-/- groups. Representative contour plots from infected sets indicate the percentages of CD45hiCD11b+ and CD45loCD11b+ cells expressing activation markers (C) MHCII, (E) CX3CR1, and (G) CD40 and the quantification are depicted in D, F, and H, respectively, comparing MI and infected WT and CD40L-/- groups. Results were expressed as mean ± SEM from 3 independent biological experiments (N = 4). *Asterisk (MI WT v/s infected WT) and #hash (infected WT v/s infected CD40L-/-) represents statistical significance calculated using unpaired Student’s t-test and Welch correction, p<0.05 was considered significant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 6.
CD40L deficiency affects T cell priming and expansion in the CLN on days 7 and 10 p.i.
On days 7 and 10 p.i., CLNs from MI and RSA59 infected (10000 PFUs) WT and CD40L-/- mice were harvested for flow cytometry analysis and stained for CD3, CD4, CD44, CD25, and Foxp3. The green color denotes WT, and blue indicates CD40L-/- mice. Primary gating was performed on singlets, followed by live cells and CD3. (A) Representative flow cytometry contour plots indicate the percentages of CD3+CD4+ T helper cells from infected sets. (B) Graphical representation of absolute numbers of CD3+CD4+ T helper cells comparing MI and infected WT and CD40L-/- groups across time points. (C) Representative flow cytometry contour plots indicating percentages of CD44+ T helper cells from infected sets. (D) Shows the absolute numbers comparing MI and infected WT and CD40L-/- groups across time points. (E) CD3+CD4+CD25+Foxp3+ T regs percentages from infected sets are represented in contour plots, and (F) absolute numbers of T regs are plotted comparing MI and infected WT and CD40L-/- groups across time points. Results were expressed as mean ± SEM from 3 independent biological experiments (N = 4). *Asterisk (black- MI WT v/s infected WT, pink- MI CD40L-/- v/s infected CD40L-/-) and #hash (infected WT v/s infected CD40L-/-) represents statistical significance calculated using unpaired Student’s t-test and Welch correction, p<0.05 was considered significant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 7.
CD40L deficiency results in severe chronic phase inflammation in RSA59 infected brains.
(A) Serial sagittal sections of brains from RSA59 infected (10000 PFUs) WT and CD40L-/- mice were analyzed for inflammation on day 30 p.i. by H&E staining and immunohistochemically by microglia/macrophage marker Iba1 and myelin protein marker PLP. The black-lined rectangles in H & E-stained sections are shown at higher magnification below the corresponding brain midsagittal sections. The blue-line rectangle in the PLP sections is further magnified to show lateral white matter tracts. Scale bars 500μm, 200μm, 20 μm. (B) Quantification of Iba1 staining intensity. The relative gene expression of (C) CD206, (D) P2Y6, and (E) TREM-2 was analyzed using qRT-PCR of RNA from day 30 p.i. brains and compared between WT and CD40L-/- mice. Results were expressed as mean ± SEM from 3 independent biological experiments (N = 5 for WT and N = 8 for CD40L-/-). *Asterisk represents statistical significance calculated using unpaired Student’s t-test and Welch correction, p<0.05 was considered significant. **p<0.01, ****p<0.0001.
Fig 8.
CD40L deficiency exacerbates chronic phase inflammation, demyelination, axon degeneration, and grey matter inflammation in the RSA59 infected spinal cord.
(A) On day 30 p.i., cross-sections of RSA59 infected (10000 PFUs) WT and CD40L-/- mouse spinal cords were analyzed for the presence of inflammatory lesions by H&E, microglia/macrophages by Iba1, demyelination by LFB, and myelin protein marker PLP by immunohistochemistry. Black boxed areas represent higher magnification of white matter area, and brown boxed areas represent higher magnification of grey matter below the corresponding spinal cord cross-sections. Blue arrows mark demyelinating plaques on the LFB-stained sections. The blue lined box in the LFB stained sections corresponds to immunohistochemical staining for PLP protein below. Scale bar 100μm, 50μm, 10μm. Quantification of (B) Iba1 staining intensity in the grey and white matter and (C) percent area of demyelination. The relative transcript abundance of (D) CD206, (E) P2Y6, and (F) TREM-2 was analyzed in the infected spinal cords using qRT-PCR and compared between WT and CD40L-/- on day 30 p.i. Results were expressed as mean ± SEM from 3 independent biological experiments (N = 5 for WT and N = 8 for CD40L-/-). *Asterisk represents statistical significance calculated using unpaired Student’s t-test and Welch correction, p<0.05 was considered significant. *p<0.05, ***p<0.001, ****p<0.0001.
Fig 9.
CD40L deficiency results in prolonged virus persistence in the CNS.
(A). The relative abundance of transcripts corresponding to the viral N gene was compared using qRT-PCR in RSA59 infected (10000 PFUs) WT and CD40L-/- mice brains (A) and spinal cord (B). (C) Anti-N immunohistochemistry revealed the in situ distribution of viral N protein in the representative brain (brain stem) and spinal cord (dorsal column) anatomical regions in WT and CD40L-/- mice on day 30 p.i. Scale bars, brain-200μm, 100μm, and spinal cord- 50μm.
Table 1.
Sequence of Primers used in the study.