Effects of Influenza Infection on Murine Alveolar Type II Cell Function THESIS Presented in Partial Fulfillment of the Requireme

Effects of Influenza Infection on Murine Alveolar Type II Cell Function

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Christian Carlisle Hofer

Graduate Program in Veterinary Biosciences

The Ohio State University

2014

Master's Examination Committee:

Ian C. Davis, DVM, PhD – Advisor

Matthew Allen, DVM, PhD

Valerie K. Bergdall, DVM, ACLAM

Estelle Cormet-Boyaka, PhD

Copyrighted by

Christian Carlisle Hofer

2014

Abstract

Influenza A virus infections result in 250,000 to 500,000 deaths annually during seasonal epidemics and pandemic outbreaks have historically killed millions. The ability of the influenza A virus genome to undergo minor changes through “antigenic drift” and major changes through “antigenic shift” poses significant challenges in developing effective annual vaccines. More importantly, these genetic alterations may yield novel strains leading to the next global pandemic. Severe respiratory disease from influenza A viruses leads to acute respiratory distress syndrome where viral replication occurs in the epithelial cells lining the alveolus. Damage to the alveolar type I (ATI) and type II (ATII) cells leads to flooding of the alveolus with edematous fluid, fibrin, erythrocytes, and other inflammatory mediators. This study investigated the effects of influenza A virus infection on those alveolar epithelial cells in a mouse model. We demonstrated that influenza infection reduces the number of ATII cells as well as dramatically alters their production of surfactant proteins. We observed a transition of ATII cells into a ATI cell phenotype by measuring the production of at ATI cell specific marker T1α/Podoplanin by

ATII cells isolated from mice post infection. Continued examination and characterization of ex-vivo isolated ATII cells can provide important information into the role of ATII cells in restoring the normal epithelial lining of a damaged alveolus. Restoration of an intact respiratory epithelium is essential in the recovery from acute lung injury.

Acknowledgments

I would like to thank Matthew Hogan, Tara Martin, Parker Woods, Famke Aeffner, and

Lisa Joseph for their assistance and technical expertise while working together in the

Davis Lab and at OSU throughout my residency. Your knowledge and friendship has been a great source of support to me over the last several years. I would also like to thank all the members of the University Laboratory Animal Resources department. Your dedication to research animal welfare and promotion of high quality science is often overlooked, but never unappreciated by me. Finally, I would like to thank the members of my MS committee: Ian Davis, Matthew Allen, Valerie Bergdall, and Estelle Cormet-

Boyaka. Your professional advice and guidance has helped develop my critical thinking and scientific writing/presentation skills which will no doubt serve me well as my career progresses.

iii

Vita

June 1987 ...... Whitewater Sr. High School

May 2001 ...... B.S. Animal Science & Zoology, University

of Florida

May 2005 ...... D.V.M., University of Florida

August 2011 to present ...... Resident Veterinarian, University

Laboratory Animal Resources, The Ohio

State University

Publications

Bowman AS, Nelson SW, Edwards JL, Hofer CC, Nolting JM, Davis IC, Slemmons RD.

2013. Comparative effectiveness of isolation techniques for contemporary Influenza A virus strains circulating in exhibition swine. J Vet Diagn Invest 25(1): 82-90.

Melendez P, Hofer CC, Donovan GA. 2006. Risk factors for udder edema and its association with lactation performance on primiparous Holstein cows in a large Florida herd, U.S.A. Prev Vet Med 76(3-4):211-21.

Landolt GA, Karasin AI, Hofer C, Mahaney J, Svaren J, Olsen CW. 2005. Use of real- time reverse transcriptase polymerase chain reaction assay and cell culture methods for detection of swine influenza A viruses. Am J Vet Res 66(1):119-24.

Fields of Study

Major Field: Veterinary Biosciences

Table of Contents

Abstract ...... ii

Vita ...... iv

Table of Contents ...... vi

List of Figures ...... ix

Chapter 1: Literature Review ...... 1

Influenza ...... 1

Influenza Epidemiology ...... 1

Influenza Virology ...... 2

Influenza Clinical Disease and Pathophysiology ...... 3

Alveolus Anatomy and Physiology ...... 4

The Alveolus...... 4

Alveolar Type I Epithelial Cells (ATI Cells) ...... 5

Alveolar Type II Epithelial Cells (ATII Cells) ...... 5

Alveolar Epithelial Cell Markers ...... 6

Pulmonary Surfactant ...... 6

Effects of Influenza A Virus on Alveolar Epithelial Cells ...... 7

Chapter 2: Study Design ...... 9

Chapter 3: Materials and Methods ...... 10

Mice ...... 10

Influenza Virus Inoculation ...... 10

Alveolar Type II Cell Isolation ...... 10

In-Vivo Imaging System (IVIS) ...... 11

Flow Cytometry...... 12

Protein Analysis by Western Blot ...... 12

Gene Expresion Analysis by rtPCR ...... 13

Other Methods ...... 13

Statistical Analysis ...... 13

Chapter 4: Results ...... 14

Effects of influenza A virus infection on C57BL/6 mice...... 14

Effects of influenza A virus infection on SP-C expression at the whole lung level ..... 14

Effects of influenza A virus infection on ATII cells ...... 14

Gene expression in isolated ATII cells following influenza A virus infection ...... 15

Protein expression in isolated ATII cells following influenza A virus infection ...... 15

Chapter 5: Discussion ...... 17

References ...... 21

vii

Appendix A: Figures ...... 32

viii

List of Figures

Figure 1. Effects of influenza A virus infection on C57BL/6 mice...... 33

Figure 2. Effects of influenza A virus infection on SP-C expression at the whole lung level...... 35

Figure 3. Effects of influenza A virus infection on ATII cells...... 36

Figure 4. Gene expression in isolated ATII cells following influenza A virus infection...... 38

Figure 5. Protein expression in isolated ATII cells following influenza A virus infection...... 39

Chapter 1: Literature Review

Influenza

Influenza Epidemiology

Influenza A virus is a respiratory pathogen that causes significant disease and deaths worldwide. Seasonal epidemics result in 3 to 5 million cases of severe illness, resulting in

250,000 to 500,000 deaths annually1. The economic impact of seasonal epidemics to the

United States alone exceeds an estimated $87 billion per year when considering direct medical costs, indirect loss of earnings, productivity, and lives2. During the twentieth century 3 pandemics have occurred: the global outbreak in 1918 to 1919 (Spanish Flu,

H1N1) which killed more than 50 million people, the Asian Flu (H2N2) in 1957 to 1958 which claimed 1 to 1.5 million lives, and the Hong Kong Flu (H3N2) in 1968 which killed over 750,000 individuals3,4. The first global pandemic of the 21st century started in the spring of 2009 and was caused by a novel swine-origin influenza A virus (designated as A (H1N1) pdm09). A recent comprehensive model estimated that fatalities from respiratory disease was 201,200 and 83,300 additional deaths were due to cardiovascular disease. In contrast to traditional seasonal epidemics, 80% of these deaths occurring in people less than 65 years of age5.

Influenza Virology

Influenza A, B, and C viruses belong to the family Orthomyxoviridae which encompasses enveloped, negative-sense, segmented RNA viruses. Epidemic influenza disease outbreaks are caused by type A and B viruses whereas type C viruses cause sporadic influenza-like disease in young children6. Type A and B genomes consist of 8 segments encoding 11 different proteins. These proteins include three transcriptases (PB1, PB2, and PA), two matrix proteins (M1 and M2), one nucleocapsid protein (NP), two non- structural proteins (NS-1 and NS-2), and two surface glycoproteins hemagglutinin (HA) and neuraminidase (NA)7. 17 different HA and 10 different NA serotypes have been identified with the recent discovery of novel influenza A viruses in South American bats8.

It is the two surface glycoproteins (HA and NA) that form the basis of strain differentiation using H and N designations (i.e. H3N2). The combination of any HA and

NA serotype is theoretically possible, however human disease is generally caused by infection with H1, H2, and H3 combined with N1 or N2 serotypes. Newly emerging strains of avian origin such as H5N1 and H7N9, have the potential to cause significant human pandemics with only minor genetic changes of the virus increasing transmissibility between people9-11.

The polymerase complex of the influenza virus has poor proofreading ability and thus point mutations often arise during replication. Consequently, minor changes can occur within the antigenic epitopes of the HA or NA glycoproteins causing “antigenic drift”.

These minor changes necessitate annual reformulation of vaccines against seasonal influenza strains. A more significant change in the genome is “antigenic shift”, which

occurs when whole genome segments are exchanged between two or more influenza A virus strains infecting the same cell. This occurs as a result of reassortment during viral packaging and can lead to devastating pandemics because of the novelty of the virus being exposed to naïve populations with no pre-existing immunity. Pigs have been described as being mixing vessels for avian and mammalian influenza viruses because they possess cellular receptors for both virus types potentially allowing infection with multiple strains of influenza A virus12. Genetic analysis of the viruses that caused the

1957 H2N2, the 1968 H1N1, and the 2009 H1N1 pandemics demonstrated that antigenic shift occurred between avian and human viruses yielding completely new strains.

Specifically, the 2009 H1N1 was derived from two previously unrelated swine-origin viruses, one of which was a derivative of the human 1918 H1N1 virus. Moreover, there appears to be consistent contributions of one or more genes from avian influenza viruses to pandemic viruses13,14.

Influenza Clinical Disease and Pathophysiology

Clinical manifestations of influenza in humans can vary from asymptomatic infection to serious illness requiring hospitalization, or death. The Centers for Disease Control and

Prevention defines influenza-like illness to include cases with one or more of the following signs and symptoms: fever >100°F (>37.8°C), muscle aches, headaches, lack of energy, dry cough, sore throat, or runny nose. Diagnosis of suspected influenza virus infection is confirmed through viral culture, serology, and several different rapid influenza diagnostic tests. The timing of accurate diagnosis of influenza A virus infection can directly impact the prognosis in some patients, as most available antiviral drugs have

shown to have limited efficacy late in the course of the disease. Therefore, it is essential that a timely and accurate diagnosis be achieved in those severe cases of influenza requiring hospitalization.

Influenza infections are usually more severe in children and the elderly15; however this varies by strain as the pandemics in 1918 and 2009 primarily affected people outside those typical age groups. Additional risk factors for contracting serious disease include underlying cardiac or other pulmonary disease, obesity, and pregnancy16-19. The primary complication of serious disease is viral pneumonia which is sometimes followed by a secondary bacterial pneumonia. Severe cases may progress to acute respiratory distress syndrome (ARDS), which is clinically characterized by cyanosis, hypoxemia, and pulmonary edema10,20-22. In influenza infections leading to ARDS, the virus extends deep into the respiratory tract and replicates within the cells lining the alveolus23. Damage to the alveolar epithelial cells can result in respiratory failure as a consequence of flooding of the alveolus with edematous fluid, fibrin, erythrocytes and other inflammatory cells preventing adequate gas exchange24. Left untreated, ARDS can be followed by multi- organ failure and ultimately death in many cases.

Alveolus Anatomy and Physiology

The Alveolus

The alveolus is the terminal portion of the mammalian lung that serves as the site of pulmonary gas exchange. Two distinct types of alveolar epithelial cells line the gas filled lumen of the alveolus. These pneumocytes lie on the basement membrane which is in contact with the endothelial cells of the pulmonary capillary beds. Pulmonary gas

exchange occurs across this epithelial-endothelial membrane. Some normal alveoli also contain resident alveolar macrophages.

Alveolar Type I Epithelial Cells (ATI Cells)

ATI cells are large, flat cells that cover approximately 95% of the surface area of the alveolus, but only account for approximately 8-15% of the total alveolar epithelia cell numbers25,26. ATI cells are terminally differentiated and are have a life span of approximately 120 days in the normal lung27.Their primary function is to allow easy gas diffusion across the epithelial-endothelial membrane yet they also have a role in fluid exchange and ion balance. There is little known about other functions of ATI cells; however, they have been shown to have a limited role in the innate immune response in bacterial pneumonia in mice28. Because they cover the majority of the lining of the alveolus, ATI cells are the primary target of many lung insults. Exposure to viral, bacterial, toxic, and even physical agents can result in ATI cell injury and death.

Alveolar Type II Epithelial Cells (ATII Cells)

ATII cells are small cuboidal cells which cover approximately 5% of the alveolar surface area not covered by the ATI cells and account for 15% of the cell numbers within the alveolus10,25. ATII cells are found in the corners of alveoli and are attached via tight junctions. They contain characteristic lamellar bodies; secretory organelles found in ATII cells and keratinocytes, and have apical microvilli. ATII cells have been extensively studied and have multiple functions. ATII cells produce, secrete, and recycle pulmonary surfactant proteins and are the primary player in fluid clearance and ion balance within

the alveoli29. Additionally, ATII cells have many innate immune functions30 and are known to differentiate into ATI cells following ATI cell injury or death31,32.

Alveolar Epithelial Cell Markers

Cellular markers exist that are either common to or unique for alveolar type 1 or type 2 cells. Use of these markers allows for specific identification of these cells types to further

33,34 35 examine cellular function and physiology. RTI40/T1α/Podoplanin and HTI56 have been identified as antigens that are unique to ATI cells. Several other antigens that are not unique to ATI cells (i.e. are expressed in other cells in the body) are not expressed in

ATII cells. Hence, they can be used to identify ATI cells within lung tissues. These ATI

36,37 + + 38 cell selective markers include Caveolin -1/-2 , Na /K - ATPase α2-isoform ,

39 40 41 Aquaporin 5 , Cytochrome P450 2B1 , and Carboxypeptidase M . ATII cell specific markers include: CD44v6, Thomsen-Friedenreich antigen, thyroid transcription factor 1,

MUC 1 42, and CD208 (DC-LAMP) 43. Additionally, ATII cells produce surfactant associated proteins SP-A, -B, -C, and –D. These proteins are selective for ATII cells; however, only SP-C is ATII cells specific30,44.

Pulmonary Surfactant

Pulmonary surfactant covers the alveolar epithelium, and is a complex mixture of phospholipids (80-85%), neutral lipids (5-10%), and a minor constituent of proteins. The primary role of surfactant is to maintain proper alveolar surface tension, which facilitates alveolar inflation during inspiration. Surfactant also provides protection against oxidants and infectious agents by regulating host defenses and immune responses45. There are four different surfactant-associated proteins: SP-A, SP-B, SP-C, and SP-D. SP-B and SP-C are

small hydrophobic proteins which are closely associated with the surfactant film. SP-B is considered to be the most important surfactant-associated protein for normal surfactant structure and function, and is essential for final packaging of surfactant within the lamellar bodies of the ATII cells46. It also regulates formation of tubular myelin47, and

SP-B deficiency is lethal in mice48,49. SP-C is the smallest surfactant-associated protein and has a transmembrane orientation. This allows SP-C to interact with the acylated phospholipid side chains which stabilizes the surfactant film50-52. SP-A and SP-D are hydrophobic molecules that belong to the family of collectins and their role is predominantly associated with host defenses and immune response53. SP-D has also been shown to influence the uptake of surfactant by ATII cells during the surfactant recycling process, which can impact the overall surfactant pool54. SP-B and SP-C are associated with and exocytosed in the lamellar bodies of ATII cells, whereas SP-A and SP-D are secreted by the ATII cells independent of lamellar body exocytosis55.

Effects of Influenza A Virus on Alveolar Epithelial Cells

Initial exposure to influenza virus in humans is typically through the upper respiratory tract and replication begins in the nasopharynx, peaking approximately 48 hours after inoculation23. In the absence of complete clearance, viral infection extends to the lower respiratory tract where the primary targets for infection and replication are the ATI and

ATII cells56. Particular Influenza virus strains may have tropism for either ATI or ATII cells, possibly as a result of differences in cell surface expression of sialosaccharides.

ATI cells generally express α-2,6-linked sialosaccharides, whereas ATII cells predominately express α-2,3-linked sialosaccharides10. Influenza A virus binds to the host

cell by attachment of the viral HA to these sialosaccharides. Autopsies from patients with fatal H5N1 infections have demonstrated that viral antigens are most prevalent in ATII cells57,58. In contrast, patients who succumbed to the 2009 pandemic H1N1 virus had viral antigens in both ATI and ATII cells. Such dual tropism is consistent with that virus being of both mammal and avian origin59. H5N1 and human-adapted H1N1 and H3N2 viruses could all replicate efficiently in human lung tissue cultures, whereas classic swine-origin viruses and low-pathology avian viruses did not and the viruses were detected nearly exclusively in ATII cells60. Although there is evidence that some influenza viruses have direct cytopathic effects on ATI cells, it is likely that cytokine- mediated destruction of these cells also contributes to the diffuse alveolar damage associated with severe influenza virus infections61. ATII cells are also targets of direct viral infection and may undergo lysis and/or apoptosis through both extrinsic and intrinsic pathways56. As previously discussed, one of the main roles of ATII cells is to serve as replacement cells for ATI cells. The mechanisms initiating and regulating this transition between phenotypes is not clearly defined. Recovery from severe influenza A virus infection starts with the regeneration of the airway epithelial cell populations and repair of the epithelial-endothelial membrane thereby restoring normal alveolar function.

Several growth factors have been shown to increase ATII cell numbers including keratinocyte growth factor (KGF), hepatocyte growth factor, and fibroblast growth factor-1062.

Chapter 2: Study Design

Gaining a better understanding of the effects and outcomes of influenza virus infection in alveolar epithelial cells may provide insight into potential therapeutic measures for treating influenza infections. Severe influenza infections lead to acute respiratory distress syndrome stemming from a breakdown of the normal alveolar epithelium and subsequent edema filling the lumen resulting in the inability to conduct normal gas exchange. The goal of this study is to isolate ATII cells from normal and influenza-infected mice in order to study the cellular changes due to influenza infection. This ex vivo approach has several benefits when compared to studies conducted using cultured ATII cells or other cell lines that do not effectively mimic the physiology of these cells in their natural environment. ATII cells isolated from mice will be characterized using multiple molecular and biochemical assays to determine how infection with influenza virus affects the function and phenotype of these cells. We hypothesize that as a consequence of the acute lung injury induced by influenza virus infection, the cellular structure and function of ATII cells will be altered and that there will be transition from the ATII cell phenotype to the ATI cell phenotype indicating an attempt to restore the normal alveolar epithelium.

Chapter 3: Materials and Methods

Mice

C57BL/6 mice were purchased from NCI (Frederick, MD) and SP-C/GFP mice on a

C57BL/6 background were bred in-house following an initial stock provided by Joe Rae

Wright (Duke University Medical Center, Durham, NC). SP-C/GFP mice express the

GFP transgene under control of the surfactant protein C (SP-C) promoter (Lo, 2008).

Mice were maintained in standard ventilated racks and provided with ad libitum food and water, as well as appropriate environmental enrichment with Nestlets™ (Ancare,

Bellmore, NY). All animal procedures were performed in compliance with local and

National Institutes of Health guidelines and were approved by The Ohio State University

Institutional Animal Care and Use Committee.

Influenza Virus Inoculation

Mice (8-12 week old) were inoculated intranasally with 10,000 plaque-forming units

(pfu) of H1N1 influenza A/WSN/33 in 50μL PBS with 0.1% BSA under ketamine- xylazine anesthesia63,64. Mice were individually marked and weighed before infection and at time of sacrifice to confirm infection status.

Alveolar Type II Cell Isolation

ATII cells were isolated from SP-C/GFP and C57BL/6 mice using a procedure previously described65. In brief, the lungs were perfused and lavaged with saline via a tracheal 10

catheter and allowed to deflate. Lungs were then inflated with approximately 2-2.5 mL of

Dispase (5 units/mL in PBS, BD Biosciences). Without allowing the lungs to completely deflate, warmed low melting point agarose (1%) was quickly instilled into the trachea

(approximately 0.3mL in volume) and the lungs were gently packed on ice for 5 minutes.

The trachea was removed at the hilus and the lungs were incubated in 5 mL of Dispase at room temperature on a rocker. Occasional mechanical agitation (shaking) of the lungs enhanced digestion of the tissues and incubation continued for 45-60 minutes until nearly all lung tissue had broken down to a slurry. DNase (0.01% in DMEM) was added to the slurry and incubated an additional 5 minutes at room temperature. The tissue slurry was passed through sequential nylon mesh (100 um, 40 um, and 21 um) and the filtrate was centrifuged. The cell pellet was re-suspended in 10mL of DMEM + 10% newborn calf serum and panned on antibody treated (anti-CD45 and anti-CD16/32, BD biosciences) petri dishes for 2 hours at 37°C and 5% CO2. Nonadherent cells were collected and re- suspended for cell counts and cytospin preps to determine purity by modified

Papanicolaou staining66.

In-Vivo Imaging System (IVIS)

SP-C/GFP mice were sacrificed using an intraperitoneal overdose of ketamine/xylazine anesthesia and their lungs were removed for determination of GFP fluorescence corresponding to SP-C production. The lungs were imaged on the IVIS 100 bioluminescent imaging workstation (Xenogen, Caliper Life Sciences). Individual lungs were imaged to measure expressed GFP fluorescence (BIN:HR(4), FOV10, f16, 2 s).

Flow Cytometry

ATII cell suspensions were fixed, permeabilized, and stained with various fluorochrome conjugated antibodies to alveolar type I cell markers (T1-α), ATII cell markers (SP-A,

SP-C, EpCAM-326), and viability dyes (Annexin-V, 7-AAD) for FACS analysis (BD

FACSCalibur and BD LSRII). ATII cell suspensions from SP-C/GFP mice were FACS sorted (FACSAria, Becton Dickson Biosciences, San Jose, CA) for high GFP signal yielding a highly enriched population of SP-C+ cells. Flow cytometry data was analyzed using FlowJo (v.10.0) software (TreeStar, Inc. Ashland, OR).

Protein Analysis by Western Blot

Whole lung homogenates were prepared in Cell Lysis Buffer (CTS #9803) using the

FastPrep®-24 bead beater instrument. Pelleted ATII cells were lysed in a small volume of lysis buffer and passed through a 30g needle to ensure complete membrane disruption.

Lysate protein concentration was determined via the BCA method (Pierce). Equal amounts of protein were loaded onto 4-12% gels and subjected to SDS-PAGE. Proteins were then transferred to pvdf membranes for antibody probing. All primary antibody incubations were carried out overnight at 4oC {Rb anti-proSPC (Millipore ab3786); Rb anti-SPA (abcam 115791); Rb anti-SPD (abcam 168366); Rb anti-H1N1 NP (GenScript

01506); Rat anti-T1α/Podoplanin (Imgenex 6743A-100); Rb anti-active Caspase3

(abcam2302); Rb anti-GAPDH (SantaCruz 25778)}. Subsequently, membranes were exposed to species-specific HRP-conjugated secondary antibodies. SuperSignal West

Pico Chemiluminescent Substrate (Pierce) allowed for the visualization of proteins.

Gene Expresion Analysis by rtPCR

RNA was isolated from ATII cell preparations using Qiagen’s RNAeasy system. Purified

RNA was converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit

(Applied Biosystems). Quantitative real-time PCR reactions were prepared using cDNA,

TaqMan® Gene Expression Master Mix, and predesigned TaqMan® probes. (sftpc Assay

ID: Mm00488144_m1; T1α/Podoplanin Assay ID: Mm01348911_g1; rn18s Assay ID:

Mm03928990_g1). Reactions were carried out on the StepOnePlus™ Real-Time PCR

System (Applied Biosystems). Relative fold-change was calculated based on the

C −ΔΔC comparative T (2 T) method.

Other Methods

Carotid arterial blood oxygen saturation, pulmonary edema by lung wet to dry weight ratio, lung homogenate viral titers, and pulmonary resistance and compliance were performed as described in earlier studies67,68.

Statistical Analysis

Descriptive statistics were calculated using Instat 3.05 (GraphPad Software, San Diego,

CA). Gaussian data distribution was verified by the method of Kolmogorov and Smirnov.

Differences between group means were analyzed by analysis of variance, with Tukey-

Kramer multiple comparison post-tests. All data are presented as mean ± SEM.

Chapter 4: Results

Effects of influenza A virus infection on C57BL/6 mice

Body weight decreases significantly by 2 days post infection (DPI) and by 6 DPI the mice have lost over 30% of their body weight (Fig. 1A). Oxygen saturation in arterial blood is significantly decreased at 6 DPI (Fig. 1B) and significant pulmonary edema is noted at both 2 and 6 DPI (Fig. 1C). Influenza viral titers from infected lungs verify influenza A virus infection without any significant alterations in the viral load between 2,

4, and 6 DPI (Fig. 1D). Additionally, total lung resistance increased significantly at 2 and

6 DPI (Fig. 1E) while compliance significantly decreased at 2 and 6 DPI (Fig. 1F).

Effects of influenza A virus infection on SP-C expression at the whole lung level

Expression of SP-C promoter genes induces expression of the GFP thereby indicating that changes in GFP correlate to changes in SP-C production. Measurement of GFP fluorescence using the IVIS demonstrates that there is a significant decrease in the level of GFP expression in whole lungs at 2 and 6 DPI when compared to uninfected lungs

(Fig. 2A & 2B).

Effects of influenza A virus infection on ATII cells

ATII cell isolates from uninfected mice demonstrate purities between 90-95% (data not shown) using Papanicolau-staining defining characteristic darkly stained granules in the cytoplasm of ATII cells (Fig. 3A). Overall yield of ATII cells is reduced at 6 DPI

compared to uninfected and at 2 DPI (Fig 3B). Freshly isolated ATII cells were analyzed by flow cytometry using the gating scheme described to select ATII cells based on

FSC/SSC properties and GFP fluorescence (Fig 3C). The use of a fluorochrome conjugated anti-SP-C antibody demonstrated the validity of using GFP fluorescence as a selection tool for SP-C producing ATII cells (Fig. 3D). Influenza A virus infection of SP-

C/GFP mice demonstrated a significant reduction in the percentage of freshly isolated

ATII cells expressing GFP (Fig. 3E) as well as a significant reduction in the mean channel fluorescence of GFP at 2 and 6 DPI (Fig. 3F).

Gene expression in isolated ATII cells following influenza A virus infection rtPCR analysis of lysates from freshly isolated ATII cells from C57BL/6 mice at 6 DPI demonstrated a significant reduction in the expression of the SP-C gene (Fig. 4A) and a significant increase in the expression of the T1α/Podoplanin gene (Fig. 4B) when compared to the expression of those genes in uninfected mice.

Protein expression in isolated ATII cells following influenza A virus infection

Expression of proSP-C proteins is notably reduced in freshly isolated ATII cells at 2 and

6 DPI as demonstrated by western blot analysis (Fig. 5A) and the ATI cell specific protein T1α/Podoplanin expression is increased at 6 DPI (Fig. 5B). Further examination of ATII cell protein expression demonstrates that the decline in production of surfactant associated proteins in not limited to SP-C, but can also be seen in SP-A and SP-D by 6

DPI. Influenza A virus NP protein was visualized in freshly isolated ATII cells at 2 and 6

DPI (Fig 5C). Freshly isolated ATII cells sorted based on GFP expression (+ or -)

demonstrate that there is a slightly higher incidence of infection in those ATII cells that are expressing SP-C as indicated by GFP fluorescence (Fig. 5D).

Chapter 5: Discussion

The use of annual vaccines and antivirals for the prevention and treatment of seasonal influenza infection fails to provide complete protection or cure disease. It is hard to predict the total impact that a significant influenza pandemic would have on society, but scenarios yielding millions of human deaths and breakdown of essential services and infrastructure are within the realm of possibility. Basic research investigating the pathogenesis of influenza viruses and the host’s cellular and immunological response to infection is critical to determine effective prophylaxis and treatment interventions. Our investigation into the effects of influenza A virus infection on murine ATII cells has demonstrated significant changes in cellular structure and function and their contribution to the pathogenesis of ARDS in severe influenza infections. Since alveolar epithelial cells are the prime target of influenza virus infection and damage to those cells result in key features of ARDS, a better understanding of the cellular mechanisms that either induce or are prompted by these changes in ATII cells populations within the diseased alveoli may yield significant advances in the development of effective influenza therapies.

We have demonstrated that ATII cells become infected with influenza A virus (FACS sort WB data) and some undergo apoptosis (Caspase WB) as a result of infection.

Cellular necrosis is another well described and accepted outcome. Our isolation method is likely too mechanically harsh to effectively capture any cells that have started to

undergo necrosis. We have also demonstrated that ATII cells undergo differentiation and begin to differentiate into ATI cells. This transition is marked by the ATII cellular expression of the T1α/Podoplanin antigen, an antigen specific to ATI cells; and the loss of SP-C or EpCAM which are both ATII cellular markers. This finding is remarkable as

EpCAM has traditionally been thought to be an antigen found on all cells of epithelial origin. Additionally, our findings have demonstrated that once a cell begins the differentiation process towards the ATI cell phenotype, the cell suspends production of

SP-C and other surfactant associated proteins. This indicates that there are likely limited cells of intermediate state that possess functional traits of both ATI and ATII cells.

Demonstration that infection with influenza A virus causes changes in surfactant protein levels has been previously described as either reducing69 or increasing70 levels of all or some surfactant associated protein levels. In fact, supplementation with artificial surfactant has been described as a potential therapeutic intervention to accompany a traditional antiviral regimen71. We have consistently demonstrated that within freshly isolated ATII cells populations SP-A, SP-C, and SP-D protein levels are reduced as a result of influenza A virus infection. Additionally, the gene transcripts for SP-C are reduced over the course of infection. Because of the varied and complex functions of

ATII in normal lung physiology, supplementation of surfactant alone is unlikely to have significant impact on the disease progression because that pathology of ARDS includes significant pulmonary edema and inflammation along with the breakdown of the normal epithelial-endothelial barrier and dysregulation of surfactant production and catabolism.

The reduction in the absolute number of ATII cells within the lung over the course of

influenza virus infection likely contributes to the breakdown in normal alveolar function and architecture. Without an adequate number of ATII cells, there is likely a reduced capability to eliminate edematous fluid from the alveoli and maintain an adequate surfactant layer required for normal alveolus inflation. Grossly, these changes can be seen in the severely congested and atelectatic lungs of mice.

Our ATII cell isolation protocol has been shown to consistently yield large number of cells of high purity. In contrast to several other studies which utilized ATII cell culture or immortalized cell lines (A549), our methodology yields cells that maintain their normal cellular structure and function. ATII cells in culture have been shown to take on non- typical shapes and display varied surface antigens that are not consistent with normal

ATII cell characteristics72. A549 cells were originally isolated and cultured from a primary lung tumor and no longer display normal ATII function. Our ex vivo isolation of

ATII cells over the course of a severe influenza A virus infection in mice uniquely demonstrated the significant changes in alveolar epithelial cell populations.

Keratinocyte growth factor (KGF) has been reported to be effective the treatment of acute lung injury from multiple causes62. It is suggested that KGF therapy has the ability to induce ATII cell hyperplasia and increase ATII cell numbers73-75. It is reasonable to deduce that KGF therapy may provide some improvement to lung pathology in influenza virus infections. We have demonstrated that in our mouse model there is a significant reduction in KGF gene expression at the whole lung level, which would seem to indicate that supplementation of KGF might have some impact; however, there is also significant reductions in the KGF receptor gene expression in ATII cells. This finding suggests that

supplementation with KGF might be ineffective; however, further scrutiny of the cellular mechanisms that could up regulate the KGF receptors may prove more productive.

Ultimately, influenza A virus infection in the mouse reduces the overall number of ATII cells within the lung. There is also a reduction in the levels of surfactant associated proteins (SP-A, SP-C, and SP-D) produced by the ATII cells and at least a fraction of the remaining ATII cells undergo differentiation to an ATI cell phenotype. A percentage of

ATII cells also exhibited signs of apoptosis. It is evident that ATII cells play a key role in the restoration of normal alveolar function. Thus, further investigation into the mechanisms that drive cellular differentiation and/or proliferation of ATII cells in the alveoli may help identify improved therapeutics for severe influenza infections.

References

1. World Health Organization. Influenza (seasonal) fact sheet no 2011. http://www.who.int/mediacentre/factsheets/fs211/en/. Updated 2014. Accessed May 23,

2014.

2. Molinari NM, Ortega-Sanchez IR, Messonnier ML, et al. The annual impact of seasonal influenza in the US: Measuring disease burden and costs. Vaccine.

2007;25(27):5086-5096.

3. Beveridge WI. The chronicle of influenza epidemics. Hist Philos Life Sci.

1991;13(2):223-234.

4. Johnson NP, Mueller J. Updating the accounts: Global mortality of the 1918-1920

"spanish" influenza pandemic. Bull Hist Med. 2002;76(1):105-115.

5. Dawood FS, Iuliano AD, Reed C, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: A modelling study. Lancet Infect Dis. 2012;12(9):687-695.

6. Matsuzaki Y, Katsushima N, Nagai Y, et al. Clinical features of influenza C virus infection in children. Journal of Infectious Diseases. 2006;193(9):1229-1235.

7. Ghedin E, Sengamalay NA, Shumway M, et al. Large-scale sequencing of human influenza reveals the dynamic nature of viral genome evolution. Nature.

2005;437(7062):1162-1166.

8. Fereidouni S, Kwasnitschka L, Balkema Buschmann A, et al. No virological evidence for an influenza A - like virus in european bats. Zoonoses and Public Health. 2014:n/a- n/a.

9. Webster RG, Peiris M, Chen H, Guan Y. H5N1 outbreaks and enzootic influenza.

Emerg Infect Dis. 2006;12(1):3-8.

10. Short KR, Kroeze EJBV, Fouchier RAM, Kuiken T. Pathogenesis of influenza- induced acute respiratory distress syndrome. The Lancet Infectious Diseases.

2014;14(1):57-69.

11. Horby P. H7N9 is a virus worth worrying about. Nature. 2013;496(7446):399.

12. Webster RG, Hulse DJ. Microbial adaptation and change: Avian influenza. Rev Sci

Tech. 2004;23(2):453-465.

13. Garten RJ, Davis CT, Russell CA, et al. Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science.

2009;325(5937):197-201.

14. Morens DM, Taubenberger JK, Fauci AS. The persistent legacy of the 1918 influenza virus. N Engl J Med. 2009;361(3):225-229. 22

15. Monto AS, Gravenstein S, Elliott M, Colopy M, Schweinle J. Clinical signs and symptoms predicting influenza infection. Arch Intern Med. 2000;160(21):3243-3247.

16. Kumar S, Chusid MJ, Willoughby RE, et al. Introduction of a novel swine-origin influenza A (H1N1) virus into milwaukee, wisconsin in 2009. Viruses. 2009;1(1):72-83.

17. Jhung MA, Swerdlow D, Olsen SJ, et al. Epidemiology of 2009 pandemic influenza

A (H1N1) in the united states. Clin Infect Dis. 2011;52 Suppl 1:S13-26.

18. Jhung MA, Davidson H, McIntyre A, et al. Preliminary results of 2009 pandemic influenza surveillance in the united states using the aggregate hospitalization and death reporting activity. Influenza Other Respir Viruses. 2011;5(5):321-327.

19. Morgan OW, Bramley A, Fowlkes A, et al. Morbid obesity as a risk factor for hospitalization and death due to 2009 pandemic influenza A(H1N1) disease. PLoS One.

2010;5(3):e9694.

20. Chotpitayasunondh T, Ungchusak K, Hanshaoworakul W, et al. Human disease from influenza A (H5N1), thailand, 2004. Emerg Infect Dis. 2005;11(2):201-209.

21. Gao R, Bhatnagar J, Blau DM, et al. Cytokine and chemokine profiles in lung tissues from fatal cases of 2009 pandemic influenza A (H1N1): Role of the host immune response in pathogenesis. Am J Pathol. 2013;183(4):1258-1268.

22. Ramsey C, Kumar A. H1N1: Viral pneumonia as a cause of acute respiratory distress syndrome. Curr Opin Crit Care. 2011;17(1):64-71. 23

23. Taubenberger JK, Morens DM. The pathology of influenza virus infections. Annu Rev

Pathol. 2008;3:499-522.

24. Piantadosi CA, Schwartz DA. The acute respiratory distress syndrome. Ann Intern

Med. 2004;141(6):460-470.

25. Crapo JD, Barry BE, Gehr P, Bachofen M, Weibel ER. Cell number and cell characteristics of the normal human lung. Am Rev Respir Dis. 1982;126(2):332-337.

26. Williams MC. Alveolar type I cells: Molecular phenotype and development. Annu

Rev Physiol. 2003;65:669-695.

27. Herzog EL, Brody AR, Colby TV, Mason R, Williams MC. Knowns and unknowns of the alveolus. Proceedings of the American Thoracic Society. 2008;5(7):778-82.

28. Yamamoto K, Ferrari JD, Cao Y, et al. Type I alveolar epithelial cells mount innate immune responses during pneumococcal pneumonia. J Immunol. 2012;189(5):2450-

2459.

29. Matthay MA, Folkesson HG, Clerici C. Lung epithelial fluid transport and the resolution of pulmonary edema. Physiol Rev. 2002;82(3):569-600.

30. Fehrenbach H. Alveolar epithelial type II cell: Defender of the alveolus revisited.

Respir Res. 2001;2(1):33-46.

31. Geiser T. Mechanisms of alveolar epithelial repair in acute lung injury--a translational approach. Swiss medical weekly. 2003;133(43-44):586-90.

32. Uhal BD. Cell cycle kinetics in the alveolar epithelium. Am J Physiol. 1997;272(6 Pt

1):L1031-45.

33. Dobbs LG, Williams MC, Gonzalez R. Monoclonal antibodies specific to apical surfaces of rat alveolar type I cells bind to surfaces of cultured, but not freshly isolated, type II cells. Biochimica et biophysica acta. 1988;970(2):146-56.

34. Gonzalez RF, Allen L, Gonzales L, Ballard PL, Dobbs LG. HTII-280, a biomarker specific to the apical plasma membrane of human lung alveolar type II cells. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

2010;58(10):891-901.

35. Dobbs LG, Gonzalez RF, Allen L, Froh DK. HTI56, an integral membrane protein specific to human alveolar type I cells. J Histochem Cytochem. 1999;47(2):129-137.

36. Kasper M, Reimann T, Hempel U, et al. Loss of caveolin expression in type I pneumocytes as an indicator of subcellular alterations during lung fibrogenesis.

Histochem Cell Biol. 1998;109(1):41-48.

37. Newman GR, Campbell L, von Ruhland C, Jasani B, Gumbleton M. Caveolin and its cellular and subcellular immunolocalisation in lung alveolar epithelium: Implications for alveolar epithelial type I cell function. Cell Tissue Res. 1999;295(1):111-120.

38. Ridge KM, Olivera WG, Saldias F, et al. Alveolar type 1 cells express the alpha2 na,K-ATPase, which contributes to lung liquid clearance. Circ Res. 2003;92(4):453-460.

39. Nielsen S, King LS, Christensen BM, Agre P. Aquaporins in complex tissues. II. subcellular distribution in respiratory and glandular tissues of rat. Am J Physiol.

1997;273(5 Pt 1):C1549-61.

40. Takahashi Y, Aida S, Suzuki E, Ito Y, Miura T, Kimula Y. Cytochrome P450 2B1 immunoreactivity in bronchiolar and alveolar epithelial cells after exposure of rats to ozone. Toxicol Appl Pharmacol. 1994;128(2):207-215.

41. Nagae A, Abe M, Becker RP, Deddish PA, Skidgel RA, Erdos EG. High concentration of carboxypeptidase M in lungs: Presence of the enzyme in alveolar type I cells. Am J Respir Cell Mol Biol. 1993;9(2):221-229.

42. Yoo SH, Jung KC, Kim JH, et al. Expression patterns of markers for type II pneumocytes in pulmonary sclerosing hemangiomas and fetal lung tissues. Arch Pathol

Lab Med. 2005;129(7):915-919.

43. Salaun B, de Saint-Vis B, Pacheco N, et al. CD208/dendritic cell-lysosomal associated membrane protein is a marker of normal and transformed type II pneumocytes.

The American Journal of Pathology. 2004;164(3):861-871.

44. Rippon HJ, Ali NN, Polak JM, Bishop AE. Initial observations on the effect of medium composition on the differentiation of murine embryonic stem cells to alveolar type II cells. Cloning Stem Cells. 2004;6(2):49-56.

45. Bernhard W, Haagsman HP, Tschernig T, et al. Conductive airway surfactant:

Surface-tension function, biochemical composition, and possible alveolar origin. Am J

Respir Cell Mol Biol. 1997;17(1):41-50.

46. Foster CD, Zhang PX, Gonzales LW, Guttentag SH. In vitro surfactant protein B deficiency inhibits lamellar body formation. Am J Respir Cell Mol Biol. 2003;29(2):259-

266.

47. Stahlman MT, Gray MP, Falconieri MW, Whitsett JA, Weaver TE. Lamellar body formation in normal and surfactant protein B-deficient fetal mice. Lab Invest.

2000;80(3):395-403.

48. Clark JC, Wert SE, Bachurski CJ, et al. Targeted disruption of the surfactant protein

B gene disrupts surfactant homeostasis, causing respiratory failure in newborn mice. Proc

Natl Acad Sci U S A. 1995;92(17):7794-7798.

49. Melton KR, Nesslein LL, Ikegami M, et al. SP-B deficiency causes respiratory failure in adult mice. Am J Physiol Lung Cell Mol Physiol. 2003;285(3):L543-9.

50. Stahlman MT, Gray MP, Falconieri MW, Whitsett JA, Weaver TE. Lamellar body formation in normal and surfactant protein B-deficient fetal mice. Lab Invest.

2000;80(3):395-403.

51. Beers MF, Mulugeta S. Surfactant protein C biosynthesis and its emerging role in conformational lung disease. Annu Rev Physiol. 2005;67:663-696.

52. Weaver TE, Conkright JJ. Function of surfactant proteins B and C. Annu Rev Physiol.

2001;63:555-578.

53. Kishore U, Greenhough TJ, Waters P, et al. Surfactant proteins SP-A and SP-D:

Structure, function and receptors. Mol Immunol. 2006;43(9):1293-1315.

54. Ikegami M. Surfactant catabolism. Respirology. 2006;11 Suppl:S24-7.

55. Rooney SA. Regulation of surfactant secretion. Comp Biochem Physiol A Mol Integr

Physiol. 2001;129(1):233-243.

56. Sanders CJ, Doherty PC, Thomas PG. Respiratory epithelial cells in innate immunity to influenza virus infection. Cell Tissue Res. 2011;343(1):13-21.

57. Nakajima N, Van Tin N, Sato Y, et al. Pathological study of archival lung tissues from five fatal cases of avian H5N1 influenza in vietnam. Mod Pathol. 2013;26(3):357-

369.

58. Uiprasertkul M, Puthavathana P, Sangsiriwut K, et al. Influenza A H5N1 replication sites in humans. Emerg Infect Dis. 2005;11(7):1036-1041.

59. Shieh WJ, Blau DM, Denison AM, et al. 2009 pandemic influenza A (H1N1):

Pathology and pathogenesis of 100 fatal cases in the united states. Am J Pathol.

2010;177(1):166-175.

60. Weinheimer VK, Becher A, Tonnies M, et al. Influenza A viruses target type II pneumocytes in the human lung. J Infect Dis. 2012;206(11):1685-1694.

61. Nicholls J,M. The battle between influenza and the innate immune response in the human respiratory tract. Infect Chemother. 2013;45(1):11-21.

62. Ware LB, Matthay MA. Keratinocyte and hepatocyte growth factors in the lung:

Roles in lung development, inflammation, and repair. Am J Physiol Lung Cell Mol

Physiol. 2002;282(5):L924-40.

63. Traylor ZP, Aeffner F, Davis IC. Influenza A H1N1 induces declines in alveolar gas exchange in mice consistent with rapid post-infection progression from acute lung injury to ARDS. Influenza Other Respir Viruses. 2013;7(3):472-479.

64. Aeffner F, Bratasz A, Flano E, Powell KA, Davis IC. Postinfection A77-1726 treatment improves cardiopulmonary function in H1N1 influenza-infected mice. Am J

Respir Cell Mol Biol. 2012;47(4):543-551.

65. Corti M, Brody AR, Harrison JH. Isolation and primary culture of murine alveolar type II cells. American journal of respiratory cell and molecular biology.

1996;14(4):309-15.

66. Dobbs LG. Isolation and culture of alveolar type II cells. Am J Physiol. 1990;258(4 Pt

1):L134-47.

67. Davis IC, Lazarowski ER, Chen FP, Hickman-Davis JM, Sullender WM, Matalon S.

Post-infection A77-1726 blocks pathophysiologic sequelae of respiratory syncytial virus infection. Am J Respir Cell Mol Biol. 2007;37(4):379-386.

68. Wolk KE, Lazarowski ER, Traylor ZP, et al. Influenza A virus inhibits alveolar fluid clearance in BALB/c mice. Am J Respir Crit Care Med. 2008;178(9):969-976.

69. Wang J, Nikrad MP, Phang T, et al. Innate immune response to influenza A virus in differentiated human alveolar type II cells. American journal of respiratory cell and molecular biology. 2011;45(3):582-91.

70. Fukushi M, Ito T, Oka T, et al. Serial histopathological examination of the lungs of mice infected with influenza A virus PR8 strain. PloS one. 2011;6(6):e21207.

71. Fukushi M, Yamashita M, Miyoshi-Akiyama T, Kubo S, Yamamoto K, Kudo K.

Laninamivir octanoate and artificial surfactant combination therapy significantly increases survival of mice infected with lethal influenza H1N1 virus. PloS one.

2012;7(8):e42419.

72. Demaio L, Tseng W, Balverde Z, et al. Characterization of mouse alveolar epithelial cell monolayers. American journal of physiology Lung cellular and molecular physiology. 2009;296(6):L1051-8.

73. Schmeckebier S, Mauritz C, Katsirntaki K, et al. Keratinocyte growth factor and dexamethasone plus elevated cAMP levels synergistically support pluripotent stem cell differentiation into alveolar epithelial type II cells. Tissue engineering Part A.

2013;19(7-8):938-51.

74. Guo J, Yi ES, Havill AM, et al. Intravenous keratinocyte growth factor protects against experimental pulmonary injury. The American journal of physiology. 1998;275(4

Pt 1):L800-5.

75. Ulich TR, Yi ES, Longmuir K, et al. Keratinocyte growth factor is a growth factor for type II pneumocytes in vivo. J Clin Invest. 1994;93(3):1298-1306.

Appendix A: Figures

Figure 1. Effects of influenza A virus infection on C57BL/6 mice.

Figure 1. Effects of influenza A virus infection on C57BL/6 mice. (A) Changes in body weight over course of infection with influenza A virus; (B) Carotid arterial oxygen saturation; (C) Measurement of pulmonary edema by lung wet:dry ratio; (D) Lung homogenate viral titers; (E) Total lung resistance; (F) Static lung compliance. (n= 6-8 mice per group, # = P < 0.005).

Figure 2. Effects of influenza A virus infection on SP-C expression at the whole lung level. (A) Representative IVIS images of whole lungs isolated from C57BL/6

(demonstrating minimal auto fluorescence in tracheal cartilage) and SPC/GFP mice (GFP fluorescence reported in photons/sec/cm2/steradian); (B) significant decreased in GFP fluorescence intensity noted at 6 DPI (n = 10/timepoint; # = P<0.0005).

Figure 3. Effects of influenza A virus infection on ATII cells.

Figure 3. Effects of influenza A virus infection on ATII cells. (A) Photomicrograph of

Papanicolau-stained ATII cells from an uninfected C57BL/6 mouse showing characteristic dark stained granules (arrows). (B) Yield of ATII cells per isolation over the course of influenza A virus infection. (C) Identification of the FSC/SSC gate used to identify ATII cells. (D) Flow cytometric scatter plot validating the use of GFP fluorescence as an indicator of SP-C production through the use of an anti-SP-C antibody conjugated with an APC flourochrome. (E) % of freshly isolated ATII cells expressing

GFP fluorescence and (F) the mean channel fluorescence (MCF) of GFP for those isolated ATII cells. (n = 4 mice / group; # = P < 0.005; ** = P < 0.0005).

Figure 4. Gene expression in isolated ATII cells following influenza A virus infection. rtPCR analysis of ATII cell lysates evaluating gene expression of (A) SP-C and (B) T1α/Podoplanin. (n = 6 mice / time point).

Figure 5. Protein expression in isolated ATII cells following influenza A virus infection.

Figure 5. Protein expression in isolated ATII cells following influenza A virus infection. WB analysis for (A) proSP-C protein; (B) T1α/T1α/Podoplanin; (C) SP-A, SP-

D, and H1N1 NP; (D) H1N1 NP on FACS GFP sorted and non-sorted ATII cells. β-Actin and GAPDH used as loading controls. (n = 3 mice / time point).