key: cord-0267164-qy0svshd
authors: Kamble, Mithila; Saadi, Fareeha; Kumar, Saurav; Saha, Bhaskar; Sarma, Jayasri Das
title: Inducible Nitric Oxide Synthase deficiency leads to early demyelination by altering the balance between pro- and anti-inflammatory responses against Murine-β-Coronavirus
date: 2022-02-10
journal: bioRxiv
DOI: 10.1101/2022.02.08.479662
sha: 1ea45843c2bdeefae46db1fb5e8813b562e31a97
doc_id: 267164
cord_uid: qy0svshd

The neurological disease Multiple sclerosis (MS) is characterized by neuroinflammation and demyelination orchestrated by the activated glial cells, the CNS infiltrating leukocytes, and their reciprocal interaction through inflammatory signals. Inducible nitric oxide synthase (iNOS), an enzyme that catalyzes sustained nitric oxide production in response to an inflammatory stimulus, is a pro-inflammatory marker expressed particularly by the microglia/macrophages (MG/Mφ) during neuroinflammation. In MS, iNOS has been reportedly associated with the disease pathology; however, studies dissecting its role in the underlying mechanisms, specifically demyelination, are limited. Therefore, we studied the role of iNOS in a recombinant beta-coronavirus-MHV-RSA59-induced neuroinflammation, which is a prototypic animal model used to investigate the pathological hallmarks of MS, neuroinflammatory demyelination, and axonal degeneration. During the acute phase of infection with RSA59, wildtype C57BL/6 (WT) mice had significantly upregulated iNOS expression in macrophages, natural killer cells, and natural killer T cells suggesting a role for iNOS in RSA59-induced neuroinflammation. Studies comparing RSA59-infected WT and iNOS-deficient mice revealed that iNOS deficiency aggravated the disease with increased CNS infiltration of macrophages and neutrophils and enhanced mortality. As early as 9-10 days after the infection, the CNS of iNOS-deficient mice had substantially higher demyelination marked with morphologically defined MG/Mφ in the demyelinating regions. Transcript analysis confirmed the significant upregulation of type2 macrophage (M2) markers-Arginase 1, CD206, and TREM2-in the CNS of iNOS-deficient mice. Corroborating to the phenotype, the iNOS-deficient mice showed a significantly higher expression of TGFβ-an anti-inflammatory cytokine- and increased T regulatory (Treg) cell infiltration, indicating an anti-inflammatory milieu established early after the infection. These observations highlight a protective role of iNOS in virus-induced neuroinflammation whereas its absence leads to MG/Mφ polarization towards a phenotype that may be involved in the exacerbated demyelination pathology. Author summary Contrary to the reported pathogenic role of inducible nitric oxide synthase (iNOS) in multiple sclerosis and related autoimmune animal models, we show that the mice deficient in iNOS show an exacerbated disease with accelerated demyelination accompanied by heightened production of an anti-inflammatory and phagocytic markers and more numbers of Tregs in a mouse model of a recombinant mouse hepatitis virus RSA59 infection. Therefore, iNOS may play protective and regulatory roles in this beta-coronavirus infection.

inhibition of iNOS or iNOS deficiency [9] [10] [11] [12] [13] [14] , while in others, it rendered the system vulnerable 77 to pathogen attack or exacerbated the disease [15] [16] [17] [18] . Though studies implicating iNOS in resulting in demyelination, which starts as early as day 5-7 p.i. and reaches its peak at day 30 p.i. 104 [26] [27] [28] [29] . 105 We previously showed an interplay between MG/Mφ and CD4+ T cells in the RSA59 induced 106 neuroinflammation model wherein the absence of CD4+ T cells impaired viral clearance from the 107 CNS and causes rare pathologies such as acute phase poliomyelitis, dorsal root ganglionic 108 inflammation, and chronic phase axonal blebbing associated with severe demyelination. This study 109 also reported that the exacerbated demyelination could result from persistent M2-like phagocytic 110 MG/Mφ observed in the CD4-/-mice, indicating a protective role of CD4+ T cells [27] . 111 Confirming evidence of the protective role of CD4+ T cells was further illustrated in a parallel 112 study performed in Ifit2-/-mice. RSA59 infected Ifit2-/-mice presented with dampened activation 7 136 anti-inflammatory cytokine and an inducer of T regulatory cells, was significantly upregulated in 137 iNOS-/-mice. In addition, iNOS-/-mice, in comparison to WT mice, showed larger demyelinating 138 plaques as early as day 9/10 p.i. populated with more amoeboid Iba1+ MG/Mφ. The marked 139 presence of amoeboid MG/Mφ in the demyelinating plaques in iNOS-/-mice was further 140 corroborated by the significant upregulation of the phagocytic marker CD206 and TREM2 and 141 anti-inflammatory M2 marker Arginase 1 at mRNA level. Combining the results, our study 142 highlighted that iNOS deficiency leads to the severity of demyelination at the acute-adaptive 143 transition phase concurrent with a robust shift towards phagocytic MG/Mφ phenotype and 144 alteration of the immune response towards an anti-inflammatory type in the RSA59 induced 145 neuroinflammatory demyelination model.

RSA59 infection induces iNOS expression in WT mice at the acute phase (day 5 p.i.).

Inflammatory stimuli are major factors that induce iNOS expression, which is otherwise strictly PFUs was analyzed by quantitative real-time PCR of brain tissue from infected WT mice at days 151 6, 10, 15, and 30 p.i. The results showed significant upregulation of iNOS mRNA at the acute 152 phase, i.e., day 6 p.i. (Figure 1 A). This coincides with the peak of the acute phase 153 neuroinflammation in WT mice, which is characterized by elevated N gene mRNA levels, viral 154 titer, and Iba1+ MG/Mφ activation as reported earlier [23, 31] . Flow cytometry further confirmed 155 the upregulation in iNOS protein expression in brain from WT mice infected at 10000 PFUs at day 156 5 p.i. iNOS+ cells were gated from the live cell population, and their numbers and median 8 158 expressing cells and the MFI for iNOS were significantly higher in the brain lysates of infected 159 WT mice as compared to the mock infected controls (Figure 1 B, C, and D). 

Together the data implied that iNOS deficiency did not affect the viral clearance in the CNS, and 208 the disease severity observed in the infected iNOS-/-mice could be due to factors intrinsic to the 209 immune responders. 

Previous studies demonstrated that RSA59 induced demyelination starts as early as day 5 p.i. and 254 reaches its peak at day 30 p.i. [19] . The status of spinal cord inflammation on both day 5 and day 255 9/10 p.i. and demyelination at day 9/10 p.i. was evaluated in WT and iNOS-/-mice infected at Thus, although pathologically no differences were observed in Iba1+ activation of brain MG/Mφ, 287 the mRNA levels of few M2-like phenotype markers were upregulated in both the brain and spinal 288 cords of iNOS-/-mice. In conclusion, in the absence of iNOS, the MG/Mφ showed an aggravated 289 anti-inflammatory phagocytic phenotype, which may contribute to increased demyelination and, 290 therefore, high disease severity in iNOS-/-mice.

In this study, we showed that intracranial infection of RSA59 in wildtype mice led to a significant 293 increase in the expression of iNOS in myeloid and lymphoid cell subsets in the brain during the 14 294 acute phase of the disease with maximum iNOS expression found in macrophages, natural killer 295 cells, and natural killer T cells. Studies carried out in iNOS-/-mice showed that despite no 296 significant differences in the viral clearance by the CNS, the absence of iNOS leads to heightened 297 disease severity and mortality. Further, iNOS-/-mice showed a significant increase in the 298 infiltration of macrophages and neutrophils in the brain at day 9/10 p.i. i.e., the acute-adaptive 299 transition phase . This phase was also marked by increased numbers of Tregs and expression of its Image analysis was performed using Fiji's basic densitometric thresholding application (Image J, 481 NIH Image, and Scion Image) as described previously [62] . Briefly, image analysis for Iba1 stained 482 sections was performed by capturing the images at the ×4 for brain and ×10 for the spinal cord so 483 that the entire section (i.e., the scan area) could be visualized within a single frame. The RGB 484 image was deconvoluted into three different colors to separate and subtract the DAB-specific 485 staining from the background hematoxylin staining. The perimeter of each brain and spinal cord 486 tissue was digitally outlined, and the area was calculated in square micrometers. A threshold value 487 was fixed for each image to ensure that all antibody-marked cells were taken into consideration.

The amounts of Iba1 staining were termed the percent area of staining.

To determine the area of demyelination, LFB stained spinal cord cross-sections from each mouse 490 were chosen and analyzed using Fiji (Image J, NIH Image, and Scion Image) [31, 62] . The total 491 perimeter of the white matter regions in each cross-section was marked, and the area was calculated 492 by adding together the dorsal, ventral, and anterior white matter areas in each section. Also, the 493 total area of the demyelinated regions was outlined and collated for each section separately. The 494 percentage of spinal cord demyelination per section per mouse was calculated.

RNA was extracted from brain tissues of RSA59 infected WT and iNOS-/-mice and mock infected 497 mice using the TRIzol isolation protocol following transcardial perfusion with 498 diethylpyrocarbonate (DEPC)-treated PBS. The total RNA concentration was measured using a 499 NanoDrop ND-2000 spectrophotometer. One microgram of RNA was used to prepare cDNA using 23 500 a high-capacity cDNA reverse transcription kit (Applied Biosystems). Quantitative real-time PCR 501 analysis was performed using a DyNAmo ColorFlash SYBR Green qPCR kit (Thermo Scientific) 502 in a Step One Plus real-time PCR system (Thermo Fisher Scientific) under the following 503 conditions: initial denaturation at 95°C for 7 min, 40 cycles of 95°C for 10 s and 60°C for 30 s and 504 melting curve analysis at 60°C for 30 s. Reactions were performed in duplicates or triplicates.

Sequences for the primers used are given in Table 1 . Absolute quantitation was achieved using the 506 threshold (ΔCT) method. mRNA expression levels of target genes in mock and RSA59 infected 507 WT and iNOS-/-mice were normalized with GAPDH and expressed as absolute mRNA expression 508 to depict the changes between the infected WT and iNOS-/-groups as well as between the mock 509 infected if any. 

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Fig 1. RSA59 infection upregulates the expression of iNOS in WT brains at the acute phase

682 15 and 30 p.i. by qRT-PCR. Results were normalized to GAPDH compared and expressed relative 683 to mock controls as Mean ± SEM (A). iNOS expression was determined by flow cytometry in 684 the brain harvested from WT mice infected at 10000 PFUs at day 5 p.i. (B) shows representative 685 flow cytometry dot plots and histograms showing percentages of iNOS+ cells, gated from live cell 686 populations that were gated from singlets, and their absolute numbers and iNOS MFI are 687 graphically represented in (C) and (D), respectively. *Asterisk represents statistical significance 688 calculated using Kruskal-Wallis test for (A) and Welch's t test for (C) and (D)

Fig 2. iNOS expression is significantly upregulated in myeloid and lymphoid immune cell

Subsequent 698 immune cell subsets were gated from iNOS+ cells and their absolute numbers and iNOS MFI were 34 699 compared between mock and infected WT groups. iNOS+ infiltrating myeloid cells were gated 700 from iNOS+ CD45hi while iNOS+ resident microglia were gated from iNOS+ CD45lo. Lymphoid 701 cell subsets expressing iNOS were gated from iNOS+ CD3+ for peripheral

Volcano plot comparing iNOS+ cells in different 707 cell subsets is represented in (G) and volcano plot comparing iNOS MFI in different cell subsets 708 is represented in (H). Results were expressed as Mean ± SEM. *Asterisk represents statistical 709 significance calculated using unpaired student's t test with Welch's correction

Fig 3 iNOS deficiency increases the disease severity in RSA59 infected mice but does not 714 affect viral replication and persistence

Clinical scores were assigned by an arbitrary scale of 0-4 as described in Materials 718 and Methods. WT is denoted by green color and iNOS-/-by pink. N gene (viral nucleocapsid gene) 719 transcripts levels in brain tissue at days 5 and 9/10p.i. (D) and in spinal cord tissue lysate at day 720 9/10 p.i. (F) were determined by qRT-PCR and represented graphically as absolute mRNA 721 expression normalized to GAPDH. (n= 3 to 5 mice per group per time point)

Statistical analysis was performed using Two-Way ANOVA with Tukey's 725 multiple comparison test for (A), (B), (D), (E), (F) and (G); and Log-rank (Mantel-Cox) test for 726 survival proportions. Results were expressed as Mean ± SEM. *Asterisk represents statistical 727 significance between infected WT and infected iNOS-/-groups

Fig 4. iNOS deficiency increases the infiltration of macrophages and neutrophils in RSA59

PFUs) brains at day 9/10 p.i. Infiltrating peripheral cells were distinguished by CD45hi 735 gating from live cell populations gated from singlets. Similarly, CD45lo gate was used to extract 736 brain resident cell populations. WT is denoted by green color and iNOS-/-by pink

Percentages of CD45hi CD11b+ Ly6G-macrophages and CD45hi CD11b+ Ly6G+ neutrophils 740 are presented in representative contour plots and quantification of their numbers in graphical form 741 in (B). (C) shows the representative contour plots of CD45lo CD11b+ microglia and its graphical 742 representation of the absolute numbers

ANOVA with Sidak's multiple comparison test between mock and infected WT and iNOS-/-744 groups. Results were expressed as Mean ± SEM. *Asterisk represents statistical significance

Fig 5. iNOS deficiency leads to increased infiltration of T regulatory cells and upregulation 749 of TGFβ mRNA expression in the brain of RSA59 infected mice at the acute-adaptive

PFUs) mice brains at day 9/10 p.i. and flow 752 cytometry analysis was performed to check lymphoid cell subsets

from CD45hi CD3+ cell populations extracted from live cells gated from singlets. WT is denoted 754 by green color and iNOS-/-by pink. (A) shows representative contour plots of CD45hi CD3+

CD4+ T cell percentages and graphical representation of their absolute numbers. Regulatory CD4+ 756 T cells were gated by CD45hi CD3+ CD4+ CD25+ FoxP3+ markers and their representative 757 contour plots are shown in with quantification of absolute numbers of Tregs are

The mRNA levels of TGFβ were determined by qRT-PCR from brain of mock and infected WT 759 and iNOS-/-brains and are represented as absolute mRNA expression normalized to GAPDH in 760 (C)

*Asterisk represents statistical significance

*p< 0.05, **p< 0.01, and ****p< 0.001. n= 3 to 4 for MI and 5 to 7 for Infected

Fig 6. iNOS deficiency led to increased demyelination with markedly high presence of 766 amoeboid microglia/ macrophages in the spinal cords of RSA59 infected mice at the acute-767 adaptive transition phase

Black boxed areas highlighted by black arrow represent higher 771 magnification of grey matter area, and brown boxed areas highlighted by brown arrow represent 772 higher magnification of white matter below the corresponding Iba1

Scale bar 100µm. (D) and (E) are the quantification of percent demyelination and percent 774

WT is denoted by green color and iNOS-/-by pink. Results were 775 expressed as Mean ± SEM. *Asterisk represents statistical significance calculated between 776 infected WT and infected iNOS-/-mice using unpaired student's t test with Welch's correction. 777 p< 0.05 was considered as significant

Fig 7. iNOS deficiency resulted in upregulation of anti-inflammatory and phagocytic 780 markers of microglia/ macrophages in RSA59 infected mice brain and spinal cords at day 781 the acute-adaptive transition phase

Transcript levels of CD206 (A and E), P2Y6 (B and F), TREM2 (C and G)

and Arg1 (D and H) were analyzed by qRT-PCR. WT is denoted by green color and iNOS-/-by 785 pink. Results were expressed as Mean ± SEM. *Asterisk represents statistical significance using 786

Two-ANOVA with Tukey's multiple comparisons test