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Viral neuroinvasionSips, George

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G.J. Sips, K. Ishizuka, A. Pekosz, H.C. Klein, D.E. Griffin, A. Sawa, J.C. Wilschut

INTERACTION OF INFLUENZA A/H1N1PDM VIRUS WITH HUMAN NASAL MUCOSA TISSUE EXPLANTS

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Manuscript in preparation.

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Interaction of influenza A/H1N1pdm virus with human nasal mucosa tissue explants

Gregorius J. Sipsa, Koko Ishizukab, Andrew Pekoszc, Hans C. Kleind, Diane E. Griffinc, Akira Sawab, and Jan C. Wilschuta* aDepartment of Medical Microbiology, Molecular Virology Section, University Medical Center Groningen, University of Groningen, The Netherlands bDepartment of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, Ma ry-land, USA cW. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hop-kins Bloomberg School of Public Health, Baltimore, Maryland, USA dDepartment of Psychiatry, University Medical Center Groningen, University of Groningen, The Netherlands *Corresponding author: Jan C. Wilschut, Department of Medical Microbiology - Molecular Virology, HPC EB88, Uni-versity Medical Center Groningen, PO Box 30.001, 9700 RB Groningen, The Netherlands. E-mail: j.c.wilschut@umcg.nl Telephone: +31 (0)50 3632733. Fax: +31 (0)50 3638171

KEYWORDSInfluenza virus, explant, upper respiratory, nasal mucosa, olfactory receptor neuron, neu-roinvasion

HIGHLIGHTSThis study (further) examines the use of cultured human nasal mucosa tissue explants to study the interaction of (neurotropic) viruses with upper respiratory mucosa target tissues and olfactory receptor neurons (ORNs)

GLOSSARYExplant: surgically-excised biopsy specimenOlfactory receptor neuron: sensory neuron involved in odor detection

ABSTRACTInfluenza is a highly prevalent respiratory virus for which extrarespiratory neurological

complications have been described. Experimental models as well as clinical evidence has indicated that the virus might infect the central nervous system (CNS) via the olfactory pathway, likely following infection of olfactory receptor neurons (ORNs) within the upper respiratory mucosa. This study assessed the interaction of influenza A/H1N1pdm virus with cultured human nasal mucosa tissue explants and analyzed whether ORNs within these

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tissue samples become infected. Nasal mucosa tissue explants were obtained from five donors (n=5) via routine surgical excision and maintained, using transwell inserts, at 37oC in a CO2 incubator (5% CO2). Explants remained intact for total culture periods of up to 96 hr as indicated by hematoxylin/eosin (H/E) staining results. Areas of influenza nucleopro-tein (NP) as well as olfactory marker protein (OMP) positivity were observed within infected explants, although no definitive infection of ORNs could, as yet, be identified in double-la-belling experiments. This study demonstrates that cultured human nasal mucosa tissue explants represent a potentially valuable tool to study the interaction of (neurotropic) viruses with upper respiratory mucosa target tissues and ORNs.

ABBREVIATIONSBSL II: biosafety level IICNS: central nervous systemDAPI: 4’,6-diamidino-2-phenylindoleDMEM: Dulbecco’s Modified Eagle MediumH/E: hematoxylin/eosinHSV1: herpes simplex virus type 1IF: immunofluorescenceMDCK: Madin-Darby canine kidneyNP: nucleoproteinOMP: olfactory marker proteinORN: olfactory receptor neuronPBS: phosphate buffered salinePVOD: post-viral olfactory dysfunctionRPMI: Roswell Park Memorial InstituteTCID50: 50% tissue-culture infectious dose

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INTRODUCTION

Influenza is a highly prevalent respiratory virus, which is associated with infections of the upper, and occasionally lower, respiratory tract. Neurological complications and/or infec-tion of the central nervous system (CNS) have, however, been described for both human as well as avian influenza viruses, including the recent influenza A/H1N1pdm virus [1-12]. Fur-thermore, links between respiratory viral infections and neurodegenerative and neuroin-flammatory disorders have been postulated [13-16].

With respect to viral invasion mechanisms, animal models have demonstrated influ-enza viruses, including H1N1 and H5N1 viruses, can infect the CNS via the olfactory route, likely following infection of olfactory receptor neurons (ORNs) within the upper respiratory mucosa [16-24].

In humans, a recent study has reported the presence of a seasonal H3N2 influenza virus in the olfactory bulb of an immunocompromised child and additionally, the attachment of H3N2, pandemic H1N1 and highly pathogenic avian influenza (HPAI) H5N1 viruses to the apical side of the human olfactory mucosa tissue has been demonstrated experimentally [25]. Furthermore, clinically, the post-viral olfactory dysfunction (PVOD) syndrome, which consists of olfactory loss and/or histological alterations in the olfactory epithelium, has been reported upon infection with respiratory viruses [26-28].

From a pathogenetic perspective, the capacity of influenza viruses to infect specific cell types, including ORNs, and spread towards the CNS, might depend upon delicate virus-host interactions involving host cell receptor expression patterns as well as viral patho-genicity determinants [20,29-31]. Several tissue-culture models have been developed to study the interactions of pathogens with cultured human tissue samples, enabling ex vivo infec-tion experiments involving viruses and natural human target tissues [32-34]. As mentioned, a recent study has experimentally demonstrated influenza viruses can attach to the apical side of human olfactory mucosa tissue. Furthermore, another study has examined the interactions of herpes simplex virus type 1 (HSV1) with cultured human nasal mucosa tissue explants [33]. Previous studies have shown ORNs might be present within, or isolated from ex vivo explants [35-38].

This study examines the interactions of influenza A/H1N1pdm virus with its natural target cells within the human upper respiratory mucosa by analyzing infection of surgically-ex-cised human nasal mucosa tissue explants and, via (co-) staining experiments, assessing whether ORNs, in this setting, become infected.

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MATERIALS AND METHODS

Explants and viruses

Human H1N1 influenza A/CA/7/2009 (A/H1N1pdm) virus was propagated and the 50% tissue-culture infectious dose (TCID50) determined in Madin-Darby canine kidney (MDCK) cells, as previously described [39,40].

Nasal mucosa tissue explants were obtained via routine surgical excision from the concha superior of multiple donors (n=5) in accordance with medical-ethical guidelines of the Johns Hopkins University (experiments were embedded within an existing study protocol of the Department of Psychiatry, Johns Hopkins University, Baltimore, Maryland, USA). Fol-lowing excision, explants were transported in transport medium, consisting of phosphate buffered saline (PBS) containing calcium and magnesium, 1 mg/mL streptomycin, 1000 U/mL penicillin (Life Technologies, Grand Island, New York, USA), 5 μg/mL fungizone (Life Technologies) and 1 mg/mL kanamycin (Life Technologies), as previously described [33].

In a biosafety level II (BSL II) setting, explants were washed using transport medium and transferred to transwell inserts, which have previously been demonstrated to be suitable for ex vivo tissue culturing purposes [32,34,41], within 24-well plates. Explants were cultured at 37°C and 5% CO2 conditions, both of which have been described previously [33]. Culture medium consisted of 50% RPMI medium, 50% Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies), 0.1 mg/mL streptomycin, 1 μg/mL penicillin and 1 μg/mL gentamycin (Life Technologies), as previously described [33]. At the end of experiments, explants were fixed using 4% paraformaldehyde and processed for further analysis within the Reference Pathology Laboratory, Johns Hopkins University, Baltimore, Maryland, USA.

Tissue-culture set up and viability of cultured explants

In a series of experiments (see Results), infected as well as uninfected control explants, cultured for 24, 48, 72, or 96 hr, were stained according to a hematoxylin/eosin (H/E) pro-tocol within the Reference Pathology Laboratory and staining results were analyzed by light microscopy using either a Nikon upright E800 microscope (Nikon, Chiyoda, Tokyo, Japan) and Image-Pro Plus software (Media Cybernetics, Rockville, Maryland, USA), a Zeiss microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) and AxioCam software (Carl Zeiss Microscopy GmbH), or an Olympus BX50 microscope (Olympus Corporation, Shin-juku, Tokyo, Japan) and cellB software (Olympus Corporation).

Infection studies

Following 24 hr of initial culturing, explants were inoculated with influenza virus within infection medium consisting of DMEM, supplemented with L-glutamine, 100 U/mL, peni-cillin, 0.1 mg/mL streptomycin and 4 μg/mL N-acetyl-trypsin. Post-inoculation, explants were cultured in culture medium for an additional 24, 48, or 72 hr (total culture periods of

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48, 72, and 96 hr, respectively), depending on the experimental set-up (see Results). Par-affin-embedded tissue sections, obtained from the Reference Pathology Laboratory, were deparaffinized using a xylene/ethanol gradient, treated with a proteinase K solution and blocked with normal goat or donkey serum or BSA. Sections were then stained with pri-mary antibodies.

To detect influenza virus, tissue sections were stained with a mouse anti-influenza nucleoprotein (NP) primary antibody [39,40]. To detect ORNs, sections were stained using a goat antibody directed against olfactory marker protein (OMP) (Wako, Chuo-ku, Osaka, Japan). Secondary antibodies consisted of AlexaFluor488 donkey anti-mouse, Alex-aFluor488 donkey anti-goat, and AlexaFluor568 donkey anti-mouse antibodies (Life Tech-nologies). Tissue sections were embedded using mounting medium containing 4’,6-dia-midino-2-phenylindole (DAPI) (Life Technologies or Vector Laboratories, Burlingame, California, USA). Staining results were analyzed by fluorescence microscopy using a Nikon upright E800 microscope and Image-Pro Plus software, or Zeiss microscope and AxioCam software. Sequential imaging was used to identify the location of infected cells. Apparent tissue auto fluorescence was addressed via imaging in multiple fluorescent channels (see Results).

RESULTS

Tissue-culture set up and viability of cultured explants

Two explants from donor 1 were allowed to incubate for 24 hr to assess the viability of explants, following initial surgical, transport, and culture procedures, at this time point. Following incubation and fixation, H/E stainings were performed. Studies were resumed according to the above described procedures following this initial “pilot” experiment.

In additional experiments (see below), viability of the explants was tested by performing H/E staining experiments on both infected and uninfected human nasal mucosa tissue explants cultured for 24, 48, or 72 hr post the initial 24 hr culture-period. Morphologically, the explants appeared to remain intact for total culture periods of up to 96 hr (Figure 1).

Infection studies

An explant from donor 2 (cut into two sections) was cultured for 24 hr. Following initial culturing, one section of the explant was inoculated with 5.5 x 105 TCID50/mL influenza virus in infection medium, whereas the other section was inoculated with infection medium without virus, for 1.5 hr. Post-inoculation, explants were cultured using culture medium for an additional 24 hr. Following culturing and fixation, explants were processed for further analysis and H/E and IF stainings performed. A cluster of NP-positive cells, located to the epithelial linings, could be seen in the infected explant (Figure 2).

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Figure 1 Viability of cultured explantsTissue sections were cultured for 24 hr, left uninfected (A-C), or infected with influenza A/H1N1pdm (D-F), and cul-tured for additional periods of time (24, 48, 72hr). Following fixation and tissue processing, H/E stainings were per-formed (total culture periods up to 96 hr). In the depicted tissue sections (donor 3), tissue integrity grossly remains intact for up to 96hr of total culturing (magnification: 200x).

Figure 2 Infection of cultured explantsTissue sections were stained with a mouse anti-NP primary antibody to assess infection with influenza A/H1N1pdm virus. An AlexaFluor488 donkey anti-mouse secondary antibody was used to detect the NP antibody and tissue sections were embedded in mounting medium containing DAPI. A positive area, located to the epithelial linings was detected in this explant (donor 2) 24 hr post-infection. Depicted are an H/E-stained (A) and corresponding immunofluorescence (IF)-stained tissue section (B/C); the asterisk in “B” indicates the approximate location of “C” (green: AlexaFluor488/NP, blue: DAPI, magnifications: H/E 200x, IF right top 100x, IF right bottom 400x).

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Explants from donors 3-5 (donor 3: two explants cut into three sections each, donors 4 and 5: one explant per donor cut into six sections each) were cultured for 24 hr. Following initial culturing, three sections per donor were inoculated with 107 TCID50/mL influenza virus in infection medium whereas the other three sections were inoculated with infection medium alone, for 1.5 hr. Post-inoculation, explants were cultured using culture medium for an additional 24, 48, or 72 hr. Following culturing and fixation, explants were processed for further analysis and H/E and IF stainings performed. Areas of OMP-positivity were observed (Figure 3)., but no definitive infection of ORNs, as assessed on the basis of double staining of individual cells, could as yet be identified, maybe due to tissue autofluorescence.

Figure 3 Presence of ORNs within cultured explantsTissue sections were stained with a goat anti-OMP primary antibody to identify ORNs. An AlexaFluor488 donkey anti-goat secondary antibody was used to detect the OMP antibody and tissue sections were embedded in mounting medium containing DAPI. The above images show monostains for OMP (donor 3) in which possible tissue autofluorescence has been addressed via imaging in the 568 channel. Depicted are AlexaFluor488 (A and D), Alex-aFluor568 (B and E) channels, as well as merged (C and F) microscopic images (green: AlexaFluor488/OMP, blue: DAPI, magnification: 200x).

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DISCUSSION

Human tissue explants represent a potentially valuable tool to study the interactions of a wide range of pathogens with their natural human target tissues under ex vivo condi-tions. Studies have recently been performed to assess the attachment patterns of influ-enza viruses to the olfactory epithelium as well as interactions of HSV1 with human nasal mucosa tissue. Furthermore, previous studies have demonstrated that ORNs might be present within, or isolated from, biopsies taken from the upper respiratory mucosa or olfactory neuroepithelium.

The current study examined the interaction of influenza A/H1N1pdm virus with cultured human nasal mucosa tissue explants. With the purpose to study the potential pathogenesis of neurological complications, we analyzed whether ORNs could be infected in this ex vivo setting. Using explants from 5 different donors (n=5), explants morphologically appeared to remain intact for total culture periods of up to 96 hr. Additionally, influenza infection, as indicated by a cluster of NP-positive cells, located to the epithelial linings, could be seen 24 hr following infection in an explant from one donor. ORNs, based upon OMP-positivity, could be identified within the cultured explants but no definite infection of these cell types was, in the currently performed experiments, identified.

The observations that the explants remained viable during the total culture period, as indicated by H/E staining results, and NP and OMP positivity demonstrated, underline the notion that this system can be used to study the potential interactions of (neurotropic) pathogens with their natural nasal mucosa target tissues and ORNs. Initially tissue sections were microscopically examined for ciliary movements to locate apical surfaces, although these movements could, under the current settings, not optically be discriminated by standard light microscopical analysis of explants upon arrival at the BSL II laboratory. Therefore, several sides of cultured explants were potentially exposed to influenza virus particles in the current tissue-culture setup. Possible future experimental options include a “physiological” orientation of surgically-excised tissue sections, enabling the inoculation of apical tissue surfaces as likely occurs upon respiratory transmission in vivo. This can be performed by pre-surgical marking of the luminal side of biopsy specimens [38] followed by fixation of tissue sections in (semi-permeable) substances such as agarose [32,42-44]. Oriented culturing and infection could then take place using agarose-embedded tissue sections cul-tured on transwell inserts, as previously described [32]. Additional options would include further assesment of the viability of nasal mucosa tissue explants cultured on transwell inserts in general, or under the above described conditions. Previously, viability of tissue explants has been determined by several methods, including TUNEL analysis [33].

In future experiments, the permissiveness of ORNs to influenza virus infection, and influ-enza receptor (i.e. sialic acid) expression patterns on cultured human nasal mucosa tissue explants and ORNs could be determined. Apart from neuroinvasion studies, such receptor

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expression patterns have been assessed on mouse ORNs [45]. Others as well as, following these earlier studies, also our group have assessed permissiveness and receptor patterns on human (neuronal) cell types in vitro, including cell types which might resemble ORNs, although ORNs were strictly speaking, not (yet) included within these studies [46-48].

Neurological complications have been described for influenza viruses, including infec-tions with influenza A/H1N1pdm virus. As recent work has shown, it seems plausible that, upon respiratory transmission, viruses come into contact with ORNs within the respiratory epithelium from where further infection might ensue, depending on a delicate interplay with immunological and anatomical parameters [17-19,49-52] In this respect, it is noteworthy that several neurodegenerative/neuroinflammatory diseases start with dysfunctioning of one or more sensory systems, such as the olfactory system [15]. Therefore, extrarespiratory CNS complications of respiratory viral infections, including their potential study in human-ized ex vivo models, deserve further attention.

ACKNOWLEDGEMENTSThe authors would like to express their sincere gratitude to all colleagues who have, in

any form, contributed to the work described. G. J. Sips was supported by a travel grant from the Graduate School of Medical Sciences (GSMS), University Medical Center Gro-ningen, University of Groningen, The Netherlands.

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