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. Author manuscript; available in PMC: 2021 Jun 1.
Published in final edited form as: Mol Cell Probes. 2020 Feb 5;51:101530. doi: 10.1016/j.mcp.2020.101530

A novel CNS-homing peptide for targeting neuroinflammatory lesions in experimental autoimmune encephalomyelitis

Bodhraj Acharya 1,2, Rakeshchandra R Meka 1,2, Shivaprasad H Venkatesha 1,2, Jason Lees 3, Tambet Teesalu 4,5, Kamal D Moudgil 1,2
PMCID: PMC7245014  NIHMSID: NIHMS1570394  PMID: 32035108

Abstract

Using phage peptide library screening, we identified peptide-encoding phages that selectively home to the inflamed central nervous system (CNS) of mice with experimental autoimmune encephalomyelitis (EAE), a model of human multiple sclerosis (MS). A phage peptide display library encoding cyclic 9-amino-acid random peptides was first screened ex-vivo for binding to the CNS tissue of EAE mice, followed by in vivo screening in the diseased mice. Phage insert sequences that were present at a higher frequency in the CNS of EAE mice than in the normal (control) mice were identified by DNA sequencing. One of the phages selected in this manner, denoted as MS-1, was shown to selectively recognize CNS tissue in EAE mice. Individually cloned phages with this insert preferentially homed to EAE CNS after an intravenous injection. Similarly, systemically administered fluorescence-labeled synthetic MS-1 peptide showed selective accumulation in the spinal cord of EAE mice. We suggest that peptide MS-1 might be useful for targeted drug delivery to CNS in EAE/MS.

Keywords: Phage library, Homing peptide, EAE, Multiple sclerosis, Probes

1. INTRODUCTION

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS) [1, 2]. The disease pathology and the underlying autoimmune processes in MS are recapitulated in experimental autoimmune encephalomyelitis (EAE), a well-studied model of the human disease [3–5]. The EAE has proven useful for the elucidation of neuroimmunological aspects of MS and in the testing of potential drug candidates/regimens for experimental therapy of MS. Currently, the treatment of MS is primarily focused on slowing the progression of the disease and there is no cure. Presently available drugs for MS have limited efficacy and their long-term use is associated with adverse reactions. These drugs for MS are given systemically, and therefore, other organs of the body besides CNS are exposed to them. Peptide ligands with preferential affinity for CNS tissue in MS could allow targeted delivery of such drugs to improve their biodistribution and to enhance their therapeutic index (benefit/risk ratio).

Using in vivo screening of phage libraries displaying random peptide sequences on their surface [6, 7], specific homing peptides for the vascular beds of several normal tissues [8] and numerous pathological lesions, including tumor vessels [9], tumor cells [10–12], tumor associated extracellular matrix [13], and arthritic synovium/vasculature [14, 15] have been described. In vivo peptide phage display is an agnostic technique with distinct advantage of the lack of a priori bias in predicting the vascular homing peptides [6, 7]. Importantly, unlike antibodies, the phage-displayed peptides typically bind to functionally important binding pockets on the target proteins and thus often possess biological activity.

A few investigators have reported the identification of homing peptides homing to the normal brain or spinal cord in mice/rats [8, 16–18]. However, there is very limited information about peptides homing to inflamed CNS in EAE/MS [19]. Therefore, the emphasis of the current study was on identifying phage-encoded peptide ligands that recognize EAE lesions, but not normal CNS. We hypothesized that inflammation and other autoimmune processes in EAE induce upregulation of biomolecules that are not expressed or are expressed at very low levels in normal CNS. The identification of peptide ligands that can bind such cellular markers would allow specific targeting of the drugs to EAE lesions in the CNS with reduced adverse reactions and systemic toxicity [20–22]. Identification of such molecules could also potentially contribute to better understanding of the disease process and provide additional targets for focused drug development efforts. We report here identification and characterization of a novel peptide denoted as MS-1 (amino acid sequence CRGGKRSSC) with tropism towards neuroinflammatory EAE lesions that is distinct from peptides previously reported to target normal mouse CNS [8, 16–18], injured CNS [23] and brain diseases other than EAE/MS [19, 24].

2. MATERIALS AND METHODS

2.1. Phage peptide-display library

A library of T7415–1b phage vectors displaying CX7C peptides, where ‘C’ is cysteine and ‘X’ is any amino acid residue, designed to display a constrained cyclic loop at the c-terminus of the p10 capsid protein, was obtained from the Institute of Biomedicine and Translational Medicine, University of Tartu (UT) (Estonia, EU). The diversity of the library was 1.28 × 109 plaque forming units (pfu). For amplification of phages for biopanning and for plaque assay, E. coli strains BLT5403 and BLT5615, respectively, were used.

2.2. Induction and evaluation of experimental autoimmune encephalomyelitis (EAE) in mice

All experiments performed on mice in this study were conducted following approval by the institutional animal care and use committee (IACUC). Myelin oligodendrocyte glycoprotein (MOG) peptide 35–55 (MOG35–55) (CS Bio, CA, USA) was emulsified with an equal volume of complete Freund’s adjuvant (CFA) containing 5 mg/ml of heat killed M. tuberculosis H37RA (Bacto, MI, USA). EAE was induced in C57BL/6 mice by subcutaneous injection of 200 μg of MOG35–55 /mouse. In addition, 2 doses of Pertussis toxin (Calbiochem, CA, USA), 400 ng each, were administered intraperitoneally at 0 and 48 h after MOG35–55 peptide injection. Thereafter, mice were observed regularly for signs of EAE, which was graded on a scale of 0 to 5 [25] (Suppl. Fig. 1): grade 1 = partial or total flaccid paralysis of tail; 2 = hind limb weakness/disrupted righting reflex; 3 = flaccid paralysis in one hind limb; 4 = flaccid paralysis in both hind limbs; and 5 = moribund/dead. The severity of disease was further confirmed by histological examination of CNS, particularly spinal cord after staining of tissue sections by hematoxylin and eosin (H&E) as well as Luxol fast blue myelin staining.

2.3. Ex-vivo and in vivo screening of phage library

For ex-vivo screening, CNS (the brain and spinal cord) tissue was harvested from normal and EAE mice after extensive perfusion of these mice with normal saline. A tissue homogenate equivalent to 100 mg of tissue was incubated with phage library (1×1011 pfu) at 4°C for 1 h (Fig. 1). Thereafter, the tissue homogenate was washed to remove the unbound phages, whereas the bound-phages were rescued using BLT5615 strain of E. coli. The phages were then amplified and 1×1011 phages were injected intravenously (i.v.) into EAE mice that had clinical score between 2 to 3. The phages were circulated for 15 min. Thereafter, animals were anesthetized, perfused with saline, and CNS tissue was collected and homogenized. The phages in that tissue were then rescued using E.coli (BLT5615).

Fig. 1. A schematic plan of ex-vivo and in vivo peptide phage library screening.

Fig. 1.

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Phages (1 × 1011) were first incubated with the CNS tissue for ex-vivo screening. The unbound phages were washed off, and the bound phages were rescued using E.coli strain BLT5403. Phages were amplified and equal concentration of phages was injected i.v. into disease-bearing EAE mice having a clinical score between 2 to 3 and control (healthy) mice. The phages were allowed to circulate for 15 min and then the animals were anesthetized, perfused, and the CNS tissue was harvested. This CNS tissue was then homogenized, the bound phages were rescued using E.coli, and the DNA samples that were prepared from them were subjected to high throughput sequencing (HTS). In parallel, in a separate group of mice, the in vivo screening cycle was repeated two more times (a total of 3 times), and the recovered phage clones were used for Sanger sequencing.

The recovered phages were amplified using E.coli BLT5403 and purified using polyethylene glycol (PEG)-NaCl as described previously [7], and then resuspended in 100 μl of PBS. Four μl of phage solution was used for PCR amplification of the peptide-encoding region of the phage genome (Qiagen HotStar Taq DNA polymerase) along with DNTP (Invitrogen, USA) and primers at 5pmol/μl (T7 up= 5’-AGCGGACCAGATTATCGCTA-3’ and T7 down= 5’-AACCCCTCAAGACCCGTTTA-3’). The correct size of PCR products was verified using agarose gel electrophoresis. The DNA concentration was measured using a Nano-drop and the samples were subjected to high throughput sequencing (HTS) as described below. For Sanger sequencing (SS), two additional rounds of in vivo screening of phage library were performed and the clones from CNS tissue from third round of screening were picked randomly. These clones were then sequenced at the Sequencing Facility of John Hopkins University, Baltimore, MD.

2.4. Amplicon high-throughput sequencing (HTS) and bioinformatics analysis

To analyze the distribution of the sequences of DNA inserts in the phage peptide library, HTS sequencing was performed employing Illumina next generation sequencing at the Core facility of the Institute for Genome Sciences (IGS) at the University of Maryland (UMB) School of Medicine. DNA libraries with molecular barcodes were constructed for sequencing on the Illumina platform using DNA Sample Prep, targeting a size of 190bp. The amplicons were directly processed for library preparation without DNA shearing step. Raw data was analyzed and the sequences were processed to trim the recognition sites and then sub-grouped at 100% identity (matching sequences). The relative percentage of sequences and the proportion of grouped sequences covered by them in EAE mice vs. normal (control) mice were assessed.

2.5. Sanger sequencing (SS) and characterization of the phage clones

After a total of 3 rounds of in vivo phage screening, the inserts of the selected phage pool were characterized as described in detail elsewhere [7, 15] - a) by PCR amplification of the phage’s insert-coding region directly from the plaque in plate; b) by sequencing the selected PCR products and identifying the sequences that appear multiple times in a particular round as well as in multiple rounds of phage selection; c) by deriving the amino acid sequence of the encoded peptide from the nucleotide sequence of the phage insert and subjecting the top-ranked sequences to motif search analysis.

The organ/tissue specificity of the individual phage clone was established by- i) examining the relative representation of peptide-encoding phages in brain/spinal cord (and in control tissues) compared to insert-less phages, and ii) by immunohistochemical staining of sections of CNS/control tissue by fluorophore-labeled anti-phage (T7) antibody, as described below.

2.6. Testing the binding of phage clones to the CNS tissue sections

The phage clones corresponding to the candidate peptide sequences were amplified using single clone amplification employing bacterial host strain E.coli BLT5403. Tissue cryosections of the brain and spinal cord were first blocked with 1% bovine serum albumin (BSA) in PBS at room temperature for 30 min, and then incubated with phage clones (1×1010) for 1 h at 4°C. Following incubation, the sections were washed and then incubated with diluted (15 ug/ml) anti -T7 rabbit antibody (UT, Estonia, EU) for 2 h at 4°C, followed by washing with PBS. The sections were then incubated with anti-rabbit antibody Alexa 549 (Cell Signaling, USA) for 1 h at 4°C. The sections were fixed with 4% paraformaldehyde (PFA), counterstained with a DNA-binding dye (Hoechst 33342) to visualize the nuclei, and mounted with Everbrite aqueous mounting medium (Biotium, USA) to protect against photobleaching.

2.7. In vivo imaging of EAE mice using Cy7-labeled peptides

Peptides were synthesized by Genscript (NJ, USA). Cyanine 7 (Cy7) dye was conjugated to the peptides (peptide MS-1 and Control peptide) at the N-terminus. The C-terminus of the peptides was not modified. For in vivo imaging, mice were anesthetized and Cy7-labeled peptide was injected to the tail vein. After 4 h, live images were taken using IVIS® Spectrum system (PerkinElmer) for florescence signals emitted from various tissues. At the termination of experiment, mice were perfused through the heart and their brain and spinal cord along with several other organs were harvested. Then ex-vivo imaging of these tissues was performed using the same equipment.

3. RESULTS

3.1. Induction of EAE, and ex-vivo and in vivo phage library screening

Mice immunized with MOG35–55 began to develop EAE within days 9–12 after the immunization. Histological examination on d18 after immunization showed mononuclear cell infiltration and demyelination in the spinal cord (Suppl. Fig. 1). Animals with a clinical score between 2 to 3 were selected for phage library screening following the experimental scheme shown in Fig. 1. One round of in vivo panning of phage library was done for subsequent high throughput screening (HTS), and 3 such rounds were performed for analysis of phage clones by manual Sanger sequencing (SS).

3.2. Phage sequencing and bioinformatics analysis

Based on HTS, the phage pool after a single in vivo selection round showed an average of ~900,000 reads. Of these, 51% contained inserts of the expected number of nucleotides. There was a selective concentration and over-representation of matched sequences in the EAE group compared to those in the normal group. This pattern suggested a more selective binding of selected phage pool to CNS tissue of EAE mice compared to normal mice. Table 1 depicts most highly represented peptides in EAE obtained by bioinformatics analysis of the HTS data. Six of these 10 sequences were also identified independently in parallel Sanger sequencing of phage clones described below.

Table 1.

Identification of CNS-homing phages after one round of in-vivo enrichment and high throughput sequencing

Sequence of phage insert Number of reads
CSLTQDQGC# 328760
CADAQADVC 140578
CKASRLGRC# 139938
CRAKGRDAC# 103044
CVGRPDA*C# 84760
CSRTEGDVC 67529
C*SSLVKTC# 61014
CKRSS*YFC# 57309
CRNGEGAQC 54832
CGEL*CNDC 54226
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#

This sequence was also identified after 3 rounds of in-vivo enrichment and Sanger sequencing

For conventional library screening, by round 3 of phage biopanning in vivo, we observed a 13-fold increase in recovered phage clones (compared to first round of in vivo biopanning) in EAE mice, while no change was seen in control mice. After Sanger sequencing (SS) of the inserts, Clustal X analysis of 43 such sequences revealed that the top enriched amino acid sequence motifs in the round 3 phage pools were: SLTQDQG at 11.6% (5/43), RAKGRDA at 6.9% (3/43), and 7 other motifs (AMGNGGD, RPGESS, PGTVKR, KRSS, GDRLV, TTPAKNN, and TEQIEER) at 4.6% (2/43) each. The sequence of six of the 10 phage peptides that contained these motifs were also identified by HTS described above. Thus, SS corroborated the results of HTS, and vice versa.

After examining the individual phage clones for their relatively higher concentration in EAE-CNS compared with normal-CNS and other organs of EAE mice, we selected 2 phage clones denoted as MS-1 (encoding peptide sequence CRGGKRSSC) and RAK (encoding peptide sequence CRAKGRDAC) for further study. Phages MS-1 and RAK encode the peptides containing the above-mentioned motifs KRSS and RAKGRDA, respectively. These two phage clones were preferred over some others such as CSLTQDQGC, which otherwise had relatively higher frequency but showed much poorer EAE versus normal discrimination than phages MS-1 and RAK.

3.3. Tissue binding and homing of selected phage clones

We next tested phages MS-1 and RAK on tissue cryosections in overlay assay. These two phages did not show any binding either to brain sections of EAE or normal mice, or to spinal cord sections of normal mice (Fig. 2 A and B). However, the MS-1 phage appeared to bind to EAE spinal cord much more avidly than RAK phage (Fig. 2B). Most of this binding of MS-1 was in the white matter region of the spinal cord compared to the gray matter (Suppl. Fig. 2).

Fig. 2. The binding of phage clones to CNS and other tissues of EAE and control mice.

Fig. 2.

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A-B: The binding of phages denoted as MS-1 (encoding peptide sequence CRGGKRSSC) and RAK (encoding peptide sequence CRAKGRDAC) to cryrosections of the brain (A) and spinal cord (B. Phage is shown in red color.) C: Biodistribution of phages MS-1 and RAK in normal (healthy) C57BL/6 (= B6) mice. Distribution of phages in tissues is shown as plaque-forming units (pfu) X 104 recovered from 100mg of tissue in this section and section ‘D’. D: Homing of phages to different organs in the EAE mice, and E: Ratio of phage MS-1/ phage RAK of different organs of EAE mice. (For C-E, phages were injected i.v. and allowed to circulate, and then recovered from tissues and characterized as described under ‘Methods’.

Both phages, when allowed to circulate following intravenous injection into normal mice, showed comparable distribution in different tissues, except for relatively higher titer of the MS-1 phage in the liver compared to the RAK phage (Fig. 2C). However, in the case of EAE mice, the MS-1 phage showed marked accumulation in the brain and the spinal cord compared to the RAK phage (Fig. 2D); the latter showed higher enrichment in the spleen over the MS-1 phage. Furthermore, homing of MS-1 to the brain and spinal cord of EAE mice was much stronger than that of RAK (Fig 2E).

Based on these comparative characteristics of MS-1 and RAK, we selected MS-1 for further testing by in vivo imaging. In view of the phage overlay results for MS-1 described above, wherein the phage showed preferential binding to the spinal cord over the brain, we focused on the spinal cord subsequent testing.

3.4. In vivo imaging of EAE mice

Cy7-labeled synthetic peptide MS-1 or a Control peptide were injected i.v. into EAE and normal (control) mice, and live imaging was performed. The results of analysis at the 4-h time point after peptide injection are shown in Fig. 3. MS-1-injected EAE mice showed much higher fluorescence signal in the spinal cord than normal mice injected with the same peptide. Furthermore, MS-1 gave a stronger signal in EAE spinal cord than the control peptide. Analysis of various tissues other than CNS of the EAE and normal mice revealed minimal to no MS-1 signal, except in the liver and kidneys, presumably because of uptake and excretion of the Cy7 dye via these two organs. There was no difference in control organ signal between the EAE and control mice.

Fig. 3. In vivo imaging using Cy7-labeled MS-1/Control peptide.

Fig. 3.

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EAE/ Normal mice were injected i.v. with Cy7-labeled MS-1 peptide or control peptide. Thereafter, in vivo imaging was performed using IVIS® Spectrum system (PerkinElmer). Results of testing at 4 h are shown (A). After that, mice were euthanized, perfused, and different organs were harvested and imaged ex vivo with IVIS® Spectrum system. The spinal cord, lung, liver, heart, spleen and kidney and blood sample in a tube, from top to bottom) are shown (B). Similar results were obtained in two other repeat experiments.

4. DISCUSSION

In organ-specific autoimmune diseases, it is increasingly recognized that unique characteristics of the target organ may contribute to the focusing of the inflammatory and autoimmune effector mechanisms to that particular organ. The main goal of our study in EAE mice was to identify peptide probes that preferentially home to the inflamed CNS and can differentiate between diseased and healthy CNS tissue. Using a combination of ex-vivo and in vivo screening of a phage-displayed peptide library, we identified a peptide denoted MS-1 that possesses tropism towards neuroinflammatory lesions. Importantly, the sequence of this peptide differs from that of the peptides previously reported to systemically home to normal [8, 16–18], injured [23], or diseased CNS [19, 24]. However, whether peptide MS-1 might also recognize other inflammatory brain pathologies remains to be studied.

The higher specificity of the MS-1 phage for EAE-CNS compared with normal-CNS is supported by higher representation of the phage-encoded peptide sequence in HTS; by in vivo enrichment of the phage clones containing that same sequence when tested by SS; by selective localization of that phage in the CNS following intravenous injection; and by homing of the dye-labeled synthetic peptide MS-1 when tested by in vivo imaging. Based on these multiple evidences, we concluded that MS-1 represents a novel CNS-homing peptide in EAE mice. Our results show that besides the MS-1 phage, the synthetic peptide encoded by this phage when injected intravenously also homes to CNS. At present, we do not know the precise mechanism involved in this transport into CNS. Collectively, the following mechanisms are among those invoked in peptide transport across BBB [26–28]: passive diffusion; active transport involving specific transporter proteins, receptor-mediated transcytosis; and shuttle proteins, many of which are ligands of certain receptors on endothelial cells (e.g., transferrin).

Short peptides such as MS-1 can be used as a ligand for targeting drugs to inflamed CNS. Cytokines and other pharmacological agents currently in use for the treatment of MS patients are given orally or parenterally and their use is associated with various side effects. We propose that the target organ-directed delivery of such therapeutic agents by using a specific CNS-homing peptide would enhance the efficacy of the treatment and help limit the side effects significantly. For this purpose, a drug of choice can be entrapped in a suitable nanoparticle, e.g., a liposome, which displays a peptide (MS-1) on its surface. (The peptide in the form of a lipopeptide is incorporated into the liposome during its preparation.) In addition, these liposomes can be surface-modified with polyethylene glycol (PEG) to avoid preferential accumulation in the reticuloendothelial system. This concept of nanoparticle-directed drug delivery has been used successfully in other diseases. For example, in previous studies in cancer, peptide-directed liposomes and iron oxide nanoworms [29, 30] have been used for drug delivery for tumors. The results of our previous study in arthritis demonstrate the feasibility of using in vivo phage peptide-display library screening to identify joint-homing peptides [15], which are promising carriers for the delivery of drugs into the inflamed joints [31]. A drug that can execute its effects locally on the cells of CNS and/or CNS-infiltrated immune cells from the periphery would be suited for targeted delivery into CNS. If that drug also has an effect on immune cells in the periphery, then CNS-targeted delivery can prevent global immune suppression. In this context, we anticipate that glucocorticoids [32], neurotrophic factors (e.g., cerebrolysin) [33], and anti-CD20 antibody (e.g., Ocrelizumab, a humanized monoclonal antibody) [34] might be better suited than some other drugs for targeted delivery into CNS. Some other potential drug candidates for consideration for targeted delivery in the near future are immunomodulatory cytokines (e.g., IL-10 and IL-27) [35–37] and inhibitors of nuclear factor-kappa B (NF-kB) and matrix metalloproteinase-9 (MMP-9) [38, 39]. This approach can later be adapted for delivery for molecular therapeutics, including modulators of specific miRNAs (e.g., miR-99b and miR-125a) that play a role in autoimmune pathogenesis of EAE/MS [40].

In-vivo imaging of EAE mice using Cy7-labeled MS-1 peptide showed marked accumulation of the fluorescence label in the spinal cord. Examination of other tissues harvested from these mice showed much weaker fluorescence signal from the liver and the kidneys, when compared with normal mice. Apparently, these two peripheral organs are in the main routes (gastrointestinal and urinary) of excretion of Cy7 from the body and therefore, some fluorescence signal intensity is expected there. However, this finding also raises the possibility that liver and kidney might be vulnerable to some side effects of the drug being targeted to CNS. The extent of side effects might depend on the drug selected for targeted delivery. Nevertheless, we believe that overall, the side effects of drug delivery using CNS-targeting peptide would be much less than that of conventional mode of oral or parenteral administration of the same drug.

We suggest that the efficacy of the above-mentioned drug delivery system in EAE/MS can be further enhanced by the use of the iRGD peptide (CRGDK/RGPDC) [41, 42] to maximize the penetration of the drug into the extravascular inflamed CNS tissue. It has been shown that iRGD peptide carrying a cryptic CendR motif can significantly enhance the penetration of ant-cancer drugs into the tumor parenchyma via neuropilin-1 in a 3-step process and thereby increase the therapeutic index of the drug [42].

Another approach for therapy involving tissue-homing peptides is their conjugation with a drug instead of preparing nanoparticles entrapping that drug. For example, a peptide homing to tumor blood vessels was coupled to an anti-cancer drug, doxorubicin [43]. This conjugate served as a more efficacious anti-cancer agent compared to the drug alone, when tested against human breast cancer tissue implanted as a xenograft in nude mice. Similarly, a conjugate of an RGD motif-containing peptide and a pro-apoptotic peptide was shown to suppress collagen-induce arthritis in mice by causing selective apoptosis of synovial neovasculature [44]. Another peptide conjugated with IL-4 was able to treat adjuvant-induced arthritis in rats [45].

Besides its use in targeted drug delivery, a CNS-homing peptide ligand can be used as a bait to identify the natural target within the CNS tissue with which this peptide interacts, as has been described for other peptides identified by phage screening [6, 7]. Such studies on our new peptide MS-1 may reveal new disease-specific, potentially druggable target molecules. We anticipate that receptors for certain mediators of neuroinflammation or neurotrophic factors might be among the likely targeted molecules for MS-1.

5. CONCLUSION

Using an innovative application of phage peptide-display library to EAE, an experimental model of human MS, our study has identified a novel peptide ligand (MS-1) that homes to inflamed CNS. This peptide can be exploited for developing targeted drug delivery to CNS to improve the efficacy and reducing the side effects of otherwise potent drugs for MS. Additional studies to identify the target of MS-1 in the CNS might lead to additional new therapeutic targets for MS.

Supplementary Material

supplemental figures

Suppl. Fig 1. Induction and evaluation of EAE in C57BL/6 mice. (a): The clinical score of EAE mice: mice with a clinical score between 2 to 3 (median score marked by an arrow) were selected for phage screening, (b): The area marked by a dashed line represents the area in the histology section shown in section ‘C’, (c): The histology of normal (control) (Left panel) and EAE mice (Right panel). The upper panel is H&E staining and the lower panel is myelin staining (10X). Areas of mononuclear cell infiltration (Top, right) and demyelination (Bottom, right) are indicated by arrowheads.

Suppl. Fig 2. The binding of phage MS-1 to spinal cord sections of EAE and normal mice. The binding of phage MS-1 to cryrosections of the spinal cord of EAE mice and normal (control) mice was tested as described in the legend to Fig. 3. Phage is shown in red color. Nuclear staining is in blue color.

1

ACKNOWLEDGEMENTS

This work was supported by 1R21NS082918 (KDM) from the National Institutes of Health (NIH), Bethesda, MD, USA. We thank Erkki Ruoslahti (Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA) for his guidance and help in planning the phage library screening and interpretation of results; and thank David W. Scott (USUHS, Bethesda, MD) for his helpful advice pertaining to the EAE model. We also thank the Institute of Genomic Sciences and the Center for Biomedical Research (CIBR) at UMB, Baltimore and the Sequencing Facility at Johns Hopkins University, Baltimore for help with sequencing of phage clones, and the Baltimore VA Medical Center for providing us with the Core facilities.

Footnotes

Conflicts of Interest: The authors declare no conflict of interest.

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Associated Data

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Supplementary Materials

supplemental figures

Suppl. Fig 1. Induction and evaluation of EAE in C57BL/6 mice. (a): The clinical score of EAE mice: mice with a clinical score between 2 to 3 (median score marked by an arrow) were selected for phage screening, (b): The area marked by a dashed line represents the area in the histology section shown in section ‘C’, (c): The histology of normal (control) (Left panel) and EAE mice (Right panel). The upper panel is H&E staining and the lower panel is myelin staining (10X). Areas of mononuclear cell infiltration (Top, right) and demyelination (Bottom, right) are indicated by arrowheads.

Suppl. Fig 2. The binding of phage MS-1 to spinal cord sections of EAE and normal mice. The binding of phage MS-1 to cryrosections of the spinal cord of EAE mice and normal (control) mice was tested as described in the legend to Fig. 3. Phage is shown in red color. Nuclear staining is in blue color.

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