Initial infectious dose dictates the innate, adaptive, and memory responses to influenza in the respiratory tract

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1 Article Initial infectious dose dictates the innate, adaptive, and memory responses to influenza in the respiratory tract Isabelle Marois, Alexandre Cloutier, Émilie Garneau, and Martin V. Richter 1 Pulmonary Division, Department of Medicine, Faculty of Medicine and Health Sciences, Université de Sherbrooke and Centre de Recherche Clinique Étienne-Le Bel, Québec, Canada RECEIVED OCTOBER 1, 11; REVISED MARCH, 1; ACCEPTED MARCH, 1. DOI: /jlb ABSTRACT Factors from the virus and the host contribute to influenza virus pathogenicity and to the development of immunity. This study thoroughly examined the effects of an initial infectious dose of virus and unveiled new findings concerning the antiviral and inflammatory responses, innate and adaptive immunity, memory responses, and protection against secondary heterologous infection. Our results demonstrated that the initial infectious dose significantly affects the gene expression of antiviral (IFN- ) and inflammatory (TNF-, IL-6, IL-1 ) cytokines and of enzymes involved in nitrosative/ oxidative stress (inos, HO-1, NQO1) early in the response to influenza. This response correlated with significantly increased recruitment of innate immune cells into the lungs of infected mice. We showed that this response also alters the subsequent accumulation of activated IFN- CD44 hi CD6L lo influenza-specific CD8 T cells into the lungs of infected mice through increased T cell-recruiting chemokine gene expression (CCL, CCL4, CCL, CXCL1). Furthermore, we demonstrated that the initial infectious dose determines the generation and the distribution of memory CD8 T cell subsets without affecting trafficking mechanisms. This impacted on immune protection against heterologous infection. Lastly, we showed that the effects on innate and adaptive immunity were not dependent on influenza strain or on the genetic background of the host. Collectively, our data show for the first time and in detail that the initial infectious dose of influenza determines the development of several aspects of antiviral immunity. This Abbreviations: ALI acute lung injury, APC allophycocyanin, CD6L CD6 ligand, EMEM MEM with Earle s salt, HAI HA inhibition assay, hi high, iemem incomplete MEM with Earle s salt, lo low, MDCK Madin Darby canine kidney, MLN mediastinal LN, NP nucleoprotein, NQO1 NAD(P)H-quinone oxidoreductase 1, PA polymerase acid, PB1- F polymerase basic 1 frame, PB polymerase basic, p.i. postinfection, PR8 A/Puerto Rico/8/4 (H1N1), qpcr quantitative PCR, RLR retinoic acid-inducible gene-like receptor, RNS reactive nitrogen species, T CM central memory T cell, T EM effector memory, TPCK tosyl phenylalanyl chloromethyl ketone, X-1 A/Hong Kong/X-1 (HN) The online version of this paper, found at includes supplemental information. study provides new insights on virus-host interaction in the generation of the global immune response to influenza. J. Leukoc. Biol. 9: 17 11; 1. Introduction Several viral factors contribute to influenza virulence in the host. These include sialic acid receptor use (preferentially,6-linked for human viruses and,-linked for avian viruses [1]), the influenza HA glycoprotein itself [, ], PB1-F, PB [4 7], and other viral factors [8, 9]. On the other hand, several host factors can also contribute to virus virulence. These factors include use of host cell proteases for HA cleavage/activation, where avian-origin viruses can be cleaved by ubiquitous pro-protein convertases, such as furin, which contributes to their pantropicity [1, 11]. Human influenza viruses are more restricted to the respiratory system, where the relevant enzymes are expressed [1, 1]. Other host factors, such as genetic susceptibility and appropriateness of the immune response, can also contribute to viral virulence [14 16]. The host must generate a proper immune response, which involves the innate and adaptive arms of immunity to successfully eliminate the virus and to limit lung damage following infection. Respiratory epithelial cells, which are the main target of influenza, detect and respond to infection by producing antiviral and inflammatory cytokines/chemokines, such as type I IFNs (IFN- and IFN- ), IL-1, IL-6, TNF-, CCL, CCL4, CXCL1, and CCL [17, 18]. This first line of defense creates an antiviral state in the epithelium and signals to innate immune cells, such as neutrophils, macrophages, DCs, and NK cells, to migrate to the site of infection to limit viral spread. In response to the virus itself or to the inflammatory mediators mentioned above, respiratory epithelial cells and innate immune cells generate oxidative molecules (ROS and RNS), which are generally counterbalanced by various antioxidant molecules and enzymes [19]. However, the combined efforts of these cell types 1. Correspondence: Pulmonary Division, Department of Medicine, Faculty of Medicine and Health Sciences, Université de Sherbrooke and Centre de Recherche Clinique Étienne-Le Bel, 1, 1e Avenue Nord, Sherbrooke, Québec, Canada, J1H N4. martin.richter@usherbrooke.ca 741-4/1/9-17 Society for Leukocyte Biology Volume 9, July 1 Journal of Leukocyte Biology 17

2 do not eliminate the virus completely. For this, the host relies on the humoral and cellular components of the adaptive immune response. To bridge between the innate and adaptive immune responses, activated DCs migrate to the draining LN of the lungs and present antigens to CD4 and to CD8 T cells [, 1]. In fact, the generation of a potent CD8 T cell response is critical for the resolution of the infection [ 6]. Following antigen recognition on DCs, specific CD8 T cells are induced to proliferate and are activated into effector cells, which migrate rapidly into the infected lungs, reaching peak infiltration 8 1 days p.i. [7 ]. Experiments with animal models have clearly shown that these virus-specific CD8 T cells are essential in the antiviral response to influenza, as delayed virus clearance and increased disease severity are observed in CD8 -deficient or CD8 -depleted mice [, 1]. Upon antigen recognition, CD8 effector T cells use several distinct effector mechanisms to eliminate invading viruses, such as the generation of antiviral mediators (IFN- and TNF- ) and direct destruction of infected cells through perforin-, granzyme-, TRAIL-, and Fas ligand-mediated mechanisms [7,, ]. During the antiviral immune response to influenza, populations of CD8 memory T cells are established in secondary lymphoid organs (T CM ) and in lung tissues (T EM ) [4 6]. These populations persist in the host and can be recalled to provide partial-to-full protection against secondary infection [4, 6, 7]. Under certain circumstances, the host s immune response can be inappropriate and contribute to the pathogenicity of influenza infection [8]. For example, high pathogenicity influenza infections have been shown to cause severe illness and elevated mortality rates in several hosts [9 4]. This outcome is linked to a dysregulation of the immune response, which leads to the elevated production of proinflammatory cytokines, often referred to as the cytokine storm or hypercytokinemia. Studies of avian HN1 and 1918 H1N1 infections have revealed that the excessive inflammatory response leads to ALI, severe respiratory distress syndrome, and multiorgan failure [4, 44]. The dysregulated immune response can be attributed to a combination of viral and host factors that interact to contribute to the pathogenesis of the disease. A high initial viral load has been linked to increased severity of infection. In part, this can be explained by an inappropriate CD8 T cell response, which contributes to viral pathogenesis [4]. However, despite the fact that the adaptive immune responses to influenza have been studied extensively in the mouse [,, 8,, 46], strikingly, little is known about the effects of initial viral load on the development of host innate, adaptive, and memory immune responses to influenza and how this influences protection against secondary infection. Some evidence suggests that the initial infectious dose can affect the development of the disease associated to influenza infection (kinetics of viral replication and time to clear the infection) and the appearance of clinical signs (morbidity as evidenced by weight loss) [47, 48]. The conclusions of these studies with regard to the immune response are sometimes contradictory and thus, difficult to integrate. Indeed, a study by Powell et. al. showed that the number of immune cells recruited to the lungs and the quantities of specific antibodies produced were independent of the infectious dose during H1N1 virus infection, although some data tended to show differences between groups [48]. According to this study, infection with a low initial infectious dose of PR8 can generate a sufficient immune response in the lungs, which could limit viral replication, accelerate viral elimination, and decrease morbidity in mice. On the other hand, a study by Hatta et al. [47], using various infectious doses of HN1 influenza, showed that infection of mice with a lethal infectious dose increased viral replication and the generation of the antiviral state without affecting the number of CD8 T cells recruited into the lungs. However, in the contraction phase, lethal infection accelerated CD8 T cell apoptosis, which suggested that this impaired T cell response could explain the delayed viral clearance and increased morbidity and mortality in infected mice [47]. These studies did not evaluate the impact of the initial infectious dose on the generation of memory T cell populations and on protective immunity during secondary infection. In this study, we sought to evaluate extensively the effects of the initial infectious dose on innate, adaptive, and memory immune responses and the potential impact on protection during secondary infection in the context of seasonal influenza infection. Globally, this study unveiled new findings about the impact of the initial infectious dose on the host immune response to influenza infection. First, we confirmed that an initial infectious dose determines lung damage, morbidity, and lung viral load during influenza infection. Secondly, we provide the first demonstration that the initial infectious dose regulates the early oxidative stress response and antiviral and inflammatory cytokine responses. This ultimately leads to a differential recruitment of innate immune cells in the lungs of infected mice. Thirdly, we show that the initial infectious dose significantly alters chemotactic signals and influences the recruitment of the influenza-specific CD8 T cells into the lungs. We demonstrated that our results were independent of mouse genetic background and viral strain. Despite significant effects on the cellular immune response, neutralizing antibody generation remained unaffected. With regard to CD8 T cell memory, we found that the initial infectious dose determines the size of CD8 T CM and T EM cell pools and that this alters the kinetics of the recall response in the lungs during a secondary heterologous infection. Finally and most importantly, in a clinical perspective, reduced immune protection against lethal secondary heterologous infection is conferred by lowdose primary infection as a result of the generation of a smaller tissue-resident CD8 T EM cell pool. MATERIALS AND METHODS Chemicals TPCK-treated trypsin from bovine pancreas and crystal violet was purchased from Sigma-Aldrich (St. Louis, MO, USA). Avicel RC-91 (microcrystalline cellulose and carboxymethycellulose sodium) was a generous gift from FMC BioPolymer (Philadelphia, PA, USA). Antibodies were purchased from ebioscience (San Diego, CA, USA) and BD Biosciences (Franklin Lakes, NJ, USA). The influenza A viral peptide epitopes NP (ASNENMETM) and PA 4 (SSLENFRAYV) were used for experiments with C7BL/6 mice and the NP (TYQRTRALV), for experiments 18 Journal of Leukocyte Biology Volume 9, July 1

3 Marois et al. Initial infectious dose dictates immunity to influenza with BALB/c mice. These peptides were purchased from AnaSpec (Fremont, CA, USA). BD Cytofix/Cytoperm kit with GolgiPlug and paraformaldehyde were, respectively, purchased from BD Biosciences and Alfa Aesar (Ward Hill, MA, USA). Trizol was purchased from Invitrogen (Burlington, ON, Canada). Cell culture MDCK cells were obtained from American Type Culture Collection (Manassas, VA, USA; CCL-4). MDCK cells were cultured using standard methods at 7 C in % CO /9% air in EMEM (Wisent, Canada), supplemented with 1% FBS, antibiotics (streptomycin 1 mg/ml and penicillin 1 U/ml), % sodium pyruvate, % nonessential amino acids, and mm L-glutamine (complete EMEM). Viruses The original stocks of mouse-adapted influenza strains, X-1 and PR8, were obtained from Dr. David Topham (University of Rochester Medical Center, New York Influenza Center of Excellence, Rochester, NY, USA). Viruses were propagated and titrated in 1-day-old embryonated hens eggs as described elsewhere [49]. Viral stock titers were determined by viral plaque assays on MDCK cells, as described below []. Infection of mice Female C7BL/6 and BALB/c mice (18 g) were purchased from Charles River Laboratories (Portage, MO, USA) and housed in specific pathogen-free conditions using microisolator technology at the Animal Care Facility of the Faculty of Medicine and Health Sciences of the Université de Sherbrooke. All experiments were approved by the Institutional Animal Ethics Committee. Mice were anesthetized by i.p. injection of Avertin (..-tribromoethanol; 4 mg/kg; Sigma-Aldrich) and infected by intranasal instillation of l PBS, containing two different infectious doses of X-1 virus (HN;. 1 or. 1 PFU/mouse) or PR8 virus (H1N1;. or PFU/mouse). Throughout the infection time-course, mouse weight was monitored to assess morbidity. A weight loss of % of the original body weight was considered as the critical limit of the experiment, and animals were euthanized according to the guidelines of the Canadian Council on Animal Care. For the secondary infection of mice, previously infected with X-1 virus, mice were anesthetized and reinfected by intranasal instillation with l PR8 virus (H1N1; 1 PFU), 6 days after primary infection. Organ isolation At indicated times p.i., mice were euthanized by injection of Avertin (7 mg/kg). The peritoneal cavity was exposed, and the spleen was removed and placed on ice in iemem containing.1% BSA instead of FBS. The trachea was cannulated, and three BALs ( 1 ml iemem) were performed to collect cells present in the airways. Lungs were purged of blood by cardiac puncture with PBS. They were removed and placed in iemem. The MLN was removed and placed in iemem. Cell isolation Spleens and MLNs were homogenized in Dounce homogenizers in iemem. Homogenized samples were filtered through a 9- m nylon mesh, centrifuged, and resuspended in iemem. Splenocyte suspensions (1 ml) were depleted of RBC using ml buffered ammonium chloride solution (Gey s solution) for min. This reaction was stopped by adding iemem. The suspension was centrifuged, resuspended in 8 ml iemem, and filtered on 7- m nylon mesh. Lung homogenates were obtained by gently rubbing organs over a -gauge wire mesh and filtered through a 9- m nylon mesh. Lung homogenates were centrifuged, and supernatants were titrated by a viral plaque assay (PFU/lungs). Lung lymphocytes were isolated at the interface of iemem and Histopaque 18 (Sigma-Aldrich) after a centrifugation of min at 8 g without the break step. Lung lymphocytes were washed once in iemem, centrifugated, and resuspended in iemem. Airway cells obtained by BAL were centrifuged and resuspended. Lysis of RBC in BAL was performed when necessary. Concentrations of all cells suspension were determined by trypan blue exclusion. Cell stimulation Aliquots of 1 6 cells were seeded in 96-well plates. Cells were stimulated in l complete EMEM containing 1 l/ml BD Golgi Plug, a protein transport inhibitor (Brefeldin A; BD Biosciences), in the presence or absence of influenza NP or PA viral peptides. Cells were incubated for 6 h at 7 C and % CO. Cells were washed with PBS-.% BSA for further staining of surface markers on adaptive immune cells. Staining of surface markers on adaptive immune cells Following stimulation, FcRs of cells were blocked with anti-cd16/ antibody (1:; BD Biosciences) for min at 4 C. Cells were then washed and labeled with primary antibodies, such as anti-cd8 PerCP-Cy. (1:4), anti-cd44 PE-Cy7 (1:8), anti-cd6l APC (1:8), for min at 4 C. After surface staining, cells were washed and resuspended in a 4% paraformaldehyde solution for fixation during 1 1 min at 4 C. Cells were kept overnight at 4 C until intracellular cytokine staining. Staining of surface markers on innate and early adaptative immune cells After blocking, cells were labeled with anti-gr1 Alexa 647 (1:6), anti- CD11b PE (1:), anti-cd11c PerCP or PE-Cy7 (1:4), and anti-f4/8 FITC or PerCP-Cy. (1:) or anti-cd8 PerCP-Cy. (1:4), anti-nk1.1 PE (1:), and anti-cd APC (1:8). These cells were fixed or analyzed immediately by flow cytometer. Intracellular cytokine staining After cell surface staining and fixation, cells were permeabilized in 1 l BD Perm/Wash buffer (BD Biosciences) for min at 4 C. Cells were centrifuged and stained for intracellular IFN- (anti-ifn- PE; 1: diluted in Perm/Wash buffer) for min at 4 C. Cells were then washed twice with Perm/Wash buffer, resuspended in PBS-.% BSA buffer, and analyzed by flow cytometry. Flow cytometry Most experimental data were collected/analyzed with FACSDiva software (BD Biosciences) using a BD FACSCanto. However, some experiments were performed using the BD FACSCalibur, and events were collected/analyzed with BD CellQuest Pro software. Postacquisition analyses were all performed using FlowJo (Tree Star, Ashland, OR, USA). Evaluation of lung damage On Day 6 p.i., the rib cage was opened, and a cannula was placed in an incision made in the trachea and tightly fixed. Warmed OCT (.8 ml) was injected slowly to inflate the lungs and held in place with sutures. Lungs were then carefully excised, and images were taken to visualize the pulmonary hemorrhage and inflammation. Viral plaque assays Assays to determine lung viral titers were performed essentially as described previously []. Two days prior to infection, MDCK cells were seeded in 4-well plates and cultured to confluence. Cells were washed twice with PBS (Wisent) and infected with l dilutions of lung homogenates in iemem for 1 h. Cells were washed once, and medium was replaced with sterile Avicel 1.8%, containing 1 g/ml of TPCK-treated trypsin (Avicel.6%, diluted 1:1 with iemem). After 48 h at 7 C % CO, cells were washed and fixed with Carnoy fixative (methanol:acetic acid, :1) for min at 4 C. Viral plaques were revealed by staining with 1% crystal violet solution in % methanol/water, 1 min. Numbers of viral plaques were counted, and lung viral titers were calculated based on the dilution factor. Volume 9, July 1 Journal of Leukocyte Biology 19

4 RNA isolation and qpcr analyses Lung homogenates ( l) were used for RNA extraction. RNA was isolated and purified using TRIzol, according to the manufacturer s protocol. RNA (1 g) was then reverse-transcribed with the Omniscript RT kit (Qiagen, Valencia, CA, USA), using random decamers (Ambion, Austin, TX, USA), followed by qpcr using the Quantitect SYBR Green PCR kit (Qiagen) with the Rotor-Gene 6 real-time PCR system (Corbett, Qiagen). qpcr reactions were completed as follows: min at 9 C, followed by 4 cycles s at 9 C, s at 7.8 C, and s at 7 C and a melting curve step. Primer sequences are detailed in Table 1. Gene expression was quantified and normalized to ribosomal 18S RNA expression using the comparative threshold method [1]. Antibody titration using the HAI Sera were recovered from mice, 6 days p.i., and were decomplemented by heating at 6 C for min. HAIs were performed, as described extensively in ref. [49]. Briefly, sera were first incubated with chicken erythrocytes to remove nonspecific agglutinating activity. Four HA units of X-1 were incubated with serial dilutions of sera and then with erythrocytes. The HAI titer for each serum sample was the reciprocal of the greatest dilution, which inhibited the agglutination of the erythrocytes completely. Statistical analyses Comparison of data from two groups of mice infected with different doses was analyzed using the unpaired Student s t test. For comparison of more than two groups, one-way ANOVA analysis, followed by Tukey post-test, was performed. Statistical analyses were done using Graphpad Prism. A P value. was considered statistically significant (P.; P.1; and P.1). RESULTS The initial infectious dose affects lung damage, morbidity, and viral replication Severe influenza infections with HN1 and 1918 pandemic H1N1 viruses can cause extensive damage to the lungs of the infected host, resulting in ALI and severe respiratory distress syndrome [4, 44]. Therefore, we assessed the effects of the initial infectious dose of seasonal influenza on the generation of lung damage in infected mice. To do so, we infected groups of C7BL/6 mice with a low dose of X-1 (HN;. 1 PFU) or a 1-fold higher dose (. 1 PFU) and compared lungs of infected mice with control (uninfected mice) at Day 6 p.i. (based on morbidity from weight loss curves). Our results showed that extensive and widespread hemorrhagic/ inflammatory areas are observed in mice infected with highdose X-1 compared with much less extensive and more restricted areas of damage in mice infected with a low dose (Fig. 1A). No damage was observed in control mice. Next, we assessed lung viral titers and morbidity in mice infected initially with either dose of X-1 over a 1-day period. Our results showed that lung viral titers peaked at Day p.i., regardless of the initial infectious dose (Fig. 1B). However, lung viral titers in mice, infected initially with high-dose X-1, reached significantly higher levels at 18 h and 48 h p.i., (61- TABLE 1. PCR Primer Sequences Primer Direction Sequence Influenza polymerase acid Forward =-CGG TCC AAA TTC CTG CTG A-= =-CAT TGG GTT CCT TCC ATC CA-= Mouse IFN- Forward =-ATG GCT AGG CTC TGT GCT TT-= =-CTC TTG TTC CTG AGG TTA T-= Mouse IFN- Forward =-AGC TCC AAG AAA GGA CGA ACA T-= =-GCC CTG TAG GTG AGG TTG ATC T-= Mouse IFN- Forward =-TCA AGT GGC ATA GAT GTG GAA GAA-= =-TGG CTC TGC AGG ATT TTC ATG-= Mouse TNF- Forward =-CCA AAG GGA TGA GAA GTT CC-= =-CTC CAC TTG GTG GTT TGC TA-= Mouse IL-1 Forward =-AAG GAG AAC CAA GCA CGA CAA AA-= =-TGG GGA ACT CTG CAG ACT CAA ACT-= Mouse IL-6 Forward =-TGA TGC ACT TGC AGA AAA CAA-= =-GGT CTT GGT CCT TAG CCA CTC-= Mouse CCL Forward =-ACT GCC TGC TGC TTC TCC TAC A-= =-AGG AAA ATG ACA CCT GGC TGG-= Mouse CCL4 Forward =-AAA CCT AAC CCC GAG CAA CA-= =-CCA TTG GTG CTG AGA ACC CT-= Mouse CCL Forward =-TCG TGC CCA CGT CAA GGA GTA TTT-= =-TCT TCT CTG GGT TGG CAC ACA CTT-= Mouse CXCL1 Forward =-GGA TGG CTG TCC TAG CTC TG-= =-TGA GCT AGG GAG GAC AAG GA-= Mouse 18S Forward =-AGG AAT TGA CGG AAG GGC AC-= =-GGA CAT CTA AGG GCA TCA CA-= Mouse inos Forward =-CAG CTG GGC TGT ACA AAC CTT C-= =-CAT TGG AAG TGA AGC GTT TCG-= Mouse NQO1 Forward =-TCA CAG GTG AGC TGA AGG AC-= =-CTT CCA GCT TCT TGT GTT CG-= Mouse HO-1 Forward =-CTG TGA ACT CTG TCC AAT G-= =-AAC TGT GTC AGG TAT CTC C-= 11 Journal of Leukocyte Biology Volume 9, July 1

5 Marois et al. Initial infectious dose dictates immunity to influenza A D Relative amplification of lung viral load Uninfected X-1,. x 1 PFU X-1,. x 1 PFU Day 6 p.i. Day 6 p.i Days post-infection B Lung viral titers (x 1 PFU/lungs) X-1,. x1 PFU X-1,. x 1 PFU Days post-infection C Percent initial weight Days post-infection Figure 1. Impact of the initial infectious dose on lung damage, viral replication, and morbidity during influenza infection. Mice were infected intranasally with a low dose (. 1 PFU; gray line) or a high dose (. 1 PFU; black line) of the X-1 virus (HN). (A) A macroscopic evaluation of lung damage was performed 6 days p.i. Dark areas indicate hemorrhagic/inflammatory areas. (B) Weight loss was monitored daily over a 1-day period following infection, and data are presented as percentage of the initial weight. (C) On indicated days, lung viral titers were evaluated by viral plaque assays. (D) The relative viral amplification was calculated as the lung viral titer, divided by the initial infectious dose. Experimental data were analyzed using Student s t test (P.; P.1; P.1). Data represent two independent experiments performed with three to four mice/experiment. and 7-fold, respectively) compared with low dose-infected mice (Fig. 1B). Viral titers returned to similar levels by Day 4 p.i. in both groups of mice. Viral titers correlated with a significant increase in morbidity, as depicted by significant weight loss, starting at Day p.i. in mice infected with high-dose X-1 (Fig. 1C). Interestingly, mice infected with the high dose of virus continued to lose weight up to Day 8 p.i., whereas mice infected with the low dose showed minimal weight loss (Fig. 1C). We also compared the relative amplification of influenza virus in the lungs of infected mice over the course of the experiment. This was done by calculating the lung viral titer at indicated time-points, divided by the initial infectious dose. Strikingly, although lung viral titers were significantly higher in mice infected with a high dose of X-1 during the early days of infection, the relative viral amplification was significantly higher (1-fold) at the peak of viral load (Day p.i.) in mice infected with the low dose of virus (Fig. 1D). Similar results were obtained with the PR8 (H1N1) strain of influenza and in BALB/c mice (see Supplemental Fig. 1). The initial infectious dose influences early inflammatory, oxidative stress, and cellular innate immune responses Influenza virus detection by intracellular sensors, such as TLRs and RLRs in epithelial cells and DCs, triggers the production of type I IFN, which induces the production of antiviral proteins within neighboring cells following ligation of type I IF- NRs [, ]. We first sought to investigate whether the initial infectious dose of virus differently influenced the early type I and II IFN responses in the respiratory tract, as we showed significant differences in viral titers and amplification in mice infected with the two infectious doses of X-1 influenza. Our results demonstrated that the expression of type I and type II IFN was induced significantly in the lungs of influenza-infected mice compared with controls (Fig. A C). Specifically, IFN- induction was -fold higher in mice that initially received a high dose of X-1 compared with low-dose infection (Fig. B). In contrast, IFN- and IFN- induction was similar in the two groups of infected mice (Fig. A and C). As inflammation contributes to morbidity in animals severely infected with influenza, we determined the induction of inflammatory cytokines at Day p.i., a time-point where initial significant differences in morbidity and peak viral titers were observed between the two groups. Gene expression of inflammatory cytokines TNF-, IL-6, and IL-1 was induced significantly in the lungs of both groups of mice compared with controls. In addition, the induction of these cytokines was significantly higher in the lungs of high dose-infected mice compared with mice that initially received a low dose of virus (TNF-, 4-fold; IL-6, 1-fold; IL-1, 4.8-fold; Fig. D F). qpcr analyses also revealed that relative gene expression of viral PA in the lungs of mice infected with high-dose X-1 was 4-fold higher than in mice infected with low-dose virus at Day p.i. (Fig. G). The activation of bronchial epithelial cells, smooth muscle cells, and lung resident macrophages by influenza infection leads to the production of NO and ROS, which are produced by various NOS and NADPH oxidases, respectively [19]. In response and in proportion to the oxidative stress, the expression of various antioxidant enzymes is increased to counteract the potential harmful effects of RNS and ROS [4]. Therefore, to evaluate oxidative stress generation, the expression of inos, NQO1, and HO-1 was assessed at different times p.i. in the lungs of infected mice. Our results showed that inos expression increased rapidly in the lungs of mice infected with highdose X-1. In fact, an early induction of inos mrna was detected at days p.i., with a peak at Day 4 p.i., returning nearly to the basal level at Days 6 p.i. In contrast, low-dose infection did not increase lung inos expression significantly compared with uninfected mice (Supplemental Fig. A). In both groups, Volume 9, July 1 Journal of Leukocyte Biology 111

6 Figure. The initial infectious dose modulates the early inflammatory responses and the innate immune responses to influenza. Type I IFNs IFN- (A) and IFN- (B) and type II IFN IFN- (C) gene expression in the lungs of mice infected with. 1 or. 1 PFU doses of X-1 was determined, 18 h p.i., by qpcr analysis using specific primers. Gene expression of TNF- (D), IL-6 (E), and IL-1 (F) and viral PA (G) was also quantified days p.i. by qpcr. Data represent n-fold changes of gene expression relative to uninfected mice after normalization to expression of ribosomal 18S RNA. (H) Innate immune cell populations were characterized in the respiratory tract (airways (BAL) and lungs) of both groups of mice by flow cytometry. Neutrophils (CD11c lo F4/8 CD11b hi Gr-1 ), NK cells (CD NK1.1 ), macrophages (F4/8 CD11c lo CD11b hi Gr-1 ), and DCs (CD11c hi CD11b hi Gr-1 ) were identified on Day p.i. (I) Early CD8 T cell infiltration into lungs (CD CD8 ) was also analyzed at Day 4 p.i. Data represent two independent experiments performed with three to four mice/infected group. One-way ANOVA analyses were performed, followed by Tukey-Kramer post-test for infected and uninfected groups (P.; P.1; P.1). Nb, Number. A B C Fold increase IFN- α Fold increase TNF- α H Nb of innate cells (x 1 ) Fold increase IL-6 Fold increase IFN - β D E F Neutrophils NK Macrophages DCs Day post-infection 18 h post-infection Fold increase IL-1β Fold increase IFN - γ Day post-infection I 4 G Fold increase PA Nb of CD8 T cells (CD CD8, x 1 ) For all panels Uninfected X-1,. x1 PFU X-1,. x1 PFU Day 4 post-infection NQO1 expression increased significantly, 18 h (.7 day) after infection, but the more severe infection induced a higher and sustained expression until Day 4 p.i. (Supplemental Fig. B). The expression of HO-1 also tended to be higher at Day p.i. (P.1) in the lungs of the mice infected with a high dose of X-1, whereas the low-dose infection did not lead to any induction of this enzyme (Supplemental Fig. C). As the initial infectious dose had profound effects on lung viral titers, inflammatory cytokines, and oxidative gene expression, we sought to determine its effects on cell populations of innate cellular immunity recruited to the site of infection. Cells from the airways (BAL) and lung parenchyma of mice infected with the two doses of X-1 were isolated at Day p.i. The number of neutrophils (CD11c lo F4/8 CD11b hi Gr-1 ), NK cells (CD NK1.1 ), macrophages (F4/8 CD11c lo CD11b hi Gr-1 ), and DCs (CD11c hi CD11b hi Gr-1 ) was determined by flow cytometry. Results from BAL and lung parenchyma were combined to show the global innate immune response at the site of infection. Recruitment of neutrophils (.9-fold), NK cells (.-fold), macrophages (1.6-fold), and DCs (-fold) was greater in mice that received the high initial infectious dose compared with the low dose of X-1 virus (Fig. H). Experiments with PR8 confirmed that these results were independent on the viral strain, as this infection induced a similar trend in the recruitment of innate immune cells, although less pronounced than during X-1 infection (Supple- 11 Journal of Leukocyte Biology Volume 9, July 1

7 Marois et al. Initial infectious dose dictates immunity to influenza mental Fig. A). In addition, we confirmed that the innate immune response to initial viral load is independent of the genetic background, as similar results were obtained using BALB/c and C7BL/6 mice (Supplemental Fig. 4A). Given that innate immune cell recruitment was largely different between groups, we also examined whether CD8 T cells could be recruited early during infection and possibly contribute to the increased morbidity observed in mice infected with the high dose of virus. Indeed, our results showed that the high infectious dose led to a -fold-greater recruitment of CD8 T cells (CD CD8 ) to the site of infection, on Day 4 p.i., compared with low-dose infection (Fig. I). The initial infectious dose alters chemotactic signals and influences the development of the adaptive immune response to influenza Given the significant impact of the initial infectious dose on antiviral and inflammatory cytokine gene expression and on innate immune cell recruitment, we next investigated its effects on the adaptive immune response. Activated, influenzaspecific CD8 T cells, which respond to influenza virus, depend on the expression of CCR and CXCR for their differential recruitment into the respiratory tract [ 7]. Therefore, we first evaluated the relative gene expression of the chemotactic signals required for CD8 T cell recruitment (namely, CCL, CCL4, and CCL; CCR ligands and CXCL1; CXCR ligand). Our results showed that at 8 days p.i., only CCL4 and CCL were increased significantly in the lungs of mice infected with low-dose X-1, whereas a trend of increase for CCL and CXCL1 expression was observed. However, in mice infected with high-dose X-1, the gene expression of all the examined chemokines was increased significantly compared with low-dose-infected mice (CCL,.4-fold; CCL4, 7.6- fold; CCL, 1.6-fold; and CXCL1,.6-fold; Fig. A D). We next examined the impact of the initial infectious dose on the recruitment of activated effector CD8 T cells (CD8 A B C D E Nb of effector CD8 T cells F Nb of virus-specific effector CD8 T cells (CD8 CD44 hi lo 4 CD6L IFN- γ, x 1 ) (CD8 CD44 hi lo CD6L, x 1 ) 4 Fold increase CCL Fold increase CCL4 Day 1 post-infection BAL Lungs MLN NP-specific Day 8 post-infection G BAL Lungs MLN BAL Lungs MLN Fold increase CCL Nb of virus-specific effector CD8 T cells (CD8 CD44 hi lo 4 CD6L IFN- γ, x 1 ) For all panels Fold increase CXCL1 PA-specific Uninfected X-1,. x1 PFU X-1,. x1 PFU Figure. Chemotactic signals and influenza-specific effector CD8 T cell recruitment into lungs are influenced by the initial infectious dose. Chemokine gene expression of CCL (A), CCL4 (B), CCL (C), and CXCL1 (D) in the lungs of mice infected with the two initial infectious doses (X-1;. 1 or. 1 PFU) was performed 8 days p.i. Data represent n- fold changes of gene expression relative to uninfected mice after normalization to expression of ribosomal 18S RNA. These data were analyzed using one-way ANOVA, followed by Tukey- Kramer post-test for infected and uninfected groups. (E) Cells from airways (BAL), lungs, and MLN were isolated from mice at Day 1 p.i., and the numbers of total effector CD8 T cells (CD8 CD44 hi CD6L lo ) in these compartments were determined. Cells were stimulated with the NP (F) or PA (G) peptides or left unstimulated for 6 h at 7 C. IFN- production was used to identify NP- or PA-specific effector CD8 T cells (CD8 CD44 hi CD6L lo IFN- ). Results in infected groups were compared and analyzed using Student s t test (P.; P.1; P.1). Results obtained with uninfected mice were presented to show the basal level of these cells. Data represent two independent experiments performed with three to four mice/experiment. Volume 9, July 1 Journal of Leukocyte Biology 11

8 CD44 hi CD6L lo ) T cells into lungs and airways of mice at Day 1 p.i. Flow cytometry analyses revealed a significantly greater recruitment of total effector CD8 T cells into the airways (BAL; 1.6-fold) and lungs (7.8-fold) of mice infected with high-dose X-1 compared with mice that received low-dose infection (Fig. E). Although higher in the group of mice that initially received a high infectious dose, the number of effector CD8 T cells in MLN was comparable with that found in mice that received a low initial dose of virus. Interestingly, similar results were obtained in mice infected with high-dose ( PFU) versus low-dose (. PFU) of PR8 virus (BAL, 16.1-fold; lungs, 6.1-fold; Supplemental Fig. B). Also, this was not dependent on host genetic background, as similar results were obtained in BALB/c mice infected with X-1 (Supplemental Fig. 4B). We determined whether the initial infectious dose influenced the recruitment of influenza-specific effector CD8 T cells to the site of infection, as the adaptive CD8 T cell response to influenza in C7BL/6 mice is dominated by two major epitopes, namely NP and PA. Our results with the X-1 virus demonstrated that IFN- NP- and PA-specific effector CD8 T cells were recruited in significantly greater numbers into the airways (BAL; NP-specific, 14.1-fold; PA-specific, 1.1- fold) and lungs (NP-specific, 1.-fold; PA-specific, 6.7-fold) of mice infected with the high dose of virus compared with lowdose infection (Fig. F and G). Our results, using the PR8 virus, demonstrated a similar trend in the number of influenzaspecific CD8 T cells recruited into the lungs and airways (BAL; Supplemental Fig. C and D). No significant differences in the numbers of effector CD8 T cells were found in the MLN of mice infected with X-1 or PR8 (Fig. E G and Supplemental Fig. B D). Similar results were obtained when BALB/c mice were infected with the two doses of X-1 virus (Supplemental Fig. 4B and C). The initial infectious dose of influenza does not influence the generation of specific antibodies against influenza virus Natural influenza virus infection or vaccination triggers a humoral response directed against influenza surface molecules, such as HA and neuraminidase, and provides protection against homologous virus and to some extent, to heterologous virus [8]. To evaluate whether the initial infectious dose affects the generation of functional antibodies, antibodies in the sera of previously infected mice (6 days p.i.) were measured using the HAI. Our results revealed that the generation of functional antibodies was not influenced by the initial infectious dose (Fig. 4A). The initial infectious dose of influenza determines the size of CD8 T CM and T EM cell pools During the primary adaptive immune response to influenza, a population of memory CD8 T cells is established [4, 6]. Two major subsets of memory cells can be distinguished: T CM (CD8 CD44 hi CD6L hi ), which reside in secondary lymphoid tissues, and T EM (CD8 CD44 hi CD6L lo ), which reside in lung tissues. Our results showed that high-dose infection induced a more prominent memory response, where tissue-resident T EM persisted at 8.1- and.1-fold-greater numbers, respectively, in the airways and lungs compared with those that received only low-dose infection (Fig. 4B). Furthermore, a 4.- fold-greater population of CD8 T CM cells can be found in the MLN of mice that were infected initially with high-dose influenza compared with low-dose infection (Fig. 4C). The initial infectious dose influences the kinetics of the recall response in the lungs during secondary heterologous infection As we found that the infectious dose affected the generation of CD8 T CM and T EM cell populations in X-1-infected mice, we determined the kinetics of virus-specific CD8 T EM cell responses in the lungs of mice following heterologous reinfection with a lethal dose of PR8 virus (H1N1; Fig. ). Sixty days after X-1 infection, memory mice were reinfected with 1 PFU of PR8, and influenza-specific memory CD8 CD44 hi CD6L lo IFN- T cell responses were evaluated on Days, 4, and 9 postsecondary infection, after stimulation with appropriate peptides. Our results demonstrated that prior to reinfection (Day ), the high initial infectious dose of X-1 tended to generate a larger population of airway- and lung-resident, virus-specific CD8 T CM cells (for NP and PA epitopes; Fig. A D). This is in agreement with results presented in Fig. 4A. At Day 4 postsecondary H1N1 infection, the number of NP- and PA-specific effector CD8 T cells reached similar levels in the airways and lungs in both groups, and on Day 9, recruitment of effector CD8 T cells to the site of infection continued to increase in both groups of mice. However, this recruitment was less in mice that had been infected initially with the higher dose of X-1 influenza (Fig. A D). We determined whether there was an impact of the initial infectious dose on the generation of virus-specific CD8 T EM cells in the MLN of mice from both initial infectious dose groups during secondary heterologous H1N1 infection. Our results showed similar numbers of NP- and PA-specific CD8 T EM cells in both groups (Fig. E and F). Reduced immune protection against lethal secondary infection is conferred by low-dose primary infection The persistence of tissue CD8 T EM cells is correlated with significant immune protection against heterologous reinfection [6, 9, 6]. As we showed that total and virus-specific tissue memory responses were different, depending on the initial infectious dose of influenza virus, we evaluated the protection conferred to mice against lethal heterologous reinfection with the PR8 virus (H1N1) and compared it with nonimmune mice infected with the same virus. At Days p.i., our results demonstrated that mice, infected initially with highdose X-1 during the primary infection, showed significantly less morbidity during lethal heterologous reinfection compared with nonimmune mice and mice infected previously with low-dose X-1 (Fig. 6A). None of the immune mice from either group died following lethal heterologous reinfection; however, mice immunized with low-dose X-1 lost a significant amount of weight ( %), up to Day 6 p.i., tracking that of 114 Journal of Leukocyte Biology Volume 9, July 1

9 Marois et al. Initial infectious dose dictates immunity to influenza A HAI titer (log 1 ) 1 X-1, X-1,. x 1 PFU. x 1 PFU B Nb of tissue effector memory CD8 T cells (CD8 CD44 hi lo CD6L, x 1 ) Day 6 post-infection BAL Uninfected X-1,. x 1 PFU X-1,. x 1 PFU Lungs C Nb of central memory CD8 T cells 4 (CD8 CD44 hi hi CD6L, x 1 ).. 1. MLN Figure 4. The initial infectious dose of influenza influences the development of CD8 T CM and T EM cell responses but not the humoral response. (A) The concentration of specific antibodies was evaluated in the serum of the two groups of mice, 6 days p.i. (X-1,. 1 or. 1 PFU). (B) CD8 T EM cells (CD8 CD44 hi CD6L lo ) were identified in BAL and lungs, 6 days p.i., whereas (C) CD8 T CM cells (CD8 CD44 hi CD6L hi ) were identified in MLN by flow cytometry. Results obtained with mice from infected groups were compared and analyzed using Student s t test (P.1; P.1). Data obtained from uninfected mice show the basal level of these cells. Results represent two independent experiments performed with three mice/experiment. nonimmune mice. Interestingly, a certain protection was conferred to these mice, as they started to recover from lethal heterologous reinfection at Day 7 and displayed significant weight gain compared with nonimmune mice (Fig. 6A). Although not statistically significant, lung viral titers tended to be lower at Day 4 postsecondary infection in mice immunized previously with high-dose X-1 compared with mice of the low dose group (Fig. 6B). DISCUSSION In this study, we thoroughly investigated the effects of the initial infectious dose on the development of antiviral, inflammatory, innate, adaptive, and memory responses in the context of seasonal influenza infection. Moreover, we examined the impact of the initial infectious dose on protective immunity against secondary heterologous infection. Interestingly, the results presented here demonstrate the importance of the initial viral load in regulating virus-host interactions that modulate immunity and protection against reinfection. We used two initial infectious doses of the influenza X-1 (HN) virus (a low dose,. 1 PFU, and a high dose,. 1 PFU) to infect C7BL/6 mice that are genetically predisposed to preferentially generate Th1-skewed responses and in which, CD8 T cells are central to viral clearance [61]. In additional experiments, we investigated the influence of the genetic background (C7BL/6 vs BALB/c) and viral strain (HN vs H1N1) in this context and demonstrated that the findings were not specific to a particular virus strain nor were they influenced by the genetics of the host. Influenza virus is detected by infected epithelial cells and DCs through intracellular PRRs, such as TLRs and RLRs, which induce the production of type I IFNs and inflammatory cytokines generating an antiviral state in neighboring cells [, ]. We found that there were significant differences in the induction of gene expression of innate antiviral and inflammatory cytokines between low- and high-dose infection. In fact, expression of IFN-, TNF-, IL-6, and IL-1 was increased significantly in mice that had received a high initial infectious dose correlating with increased lung damage. This early induction of antiviral and inflammatory cytokines during influenza infection is characteristic of viral infections in humans and mice [8, 6, 6]. Our results showed that a high infectious dose provides stronger antiviral and inflammatory signals, which contribute to increased recruitment of innate and adaptive immune cells into the respiratory tract early after infection. These strong inflammatory signals correlated with higher lung viral titers and increased morbidity in mice infected initially with the high infectious dose. The host immune response contributed to viral pathogenicity in mice, infected initially with the high dose of X-1, as lung viral titers were similar in both groups by Day 4 p.i., and there was a significant difference in morbidity between groups from Day 4 to Day 8 p.i. Acute inflammatory cell recruitment is necessary to contain influenza infection, but on the other hand, it also contributes to influenza pathogenicity. Among innate immune cells, NK cells have been shown to participate in influenza virus control, and NK cell depletion prior to infection increases susceptibility of mice to influenza-induced morbidity and mortality [64, 6]. In addition, influenza infection leads to a significant recruitment of monocytes that differentiate into macrophages, which produce large amounts of inflammatory cytokines, such as IL-6 and TNF- [66]. This is in perfect agreement with our observations in mice infected with high-dose influenza, where greater morbidity correlated with the infiltration of larger numbers of innate immune cells, such as macrophages, and with induction of inflammatory cytokines. Depletion of macrophages, prior to influenza infection, results in an uncontrolled replication of the virus and in increased mortality [67]. This indicates that macrophages play an essential role in the control of influenza virus replication at early time-points of infection. Thus, massive recruitment of macrophages at Day p.i. can account for control of viral titers and their subsequent reduction by Day 4 p.i. In addition, neutrophils have been linked to the initial control of influenza infection [67], but their Volume 9, July 1 Journal of Leukocyte Biology 11

10 A 6. x 1. x 1. x 1 4 NP-specific BAL B 4.1 x 1.1 x 1 1. x 1 4 PA-specific BAL Figure. Recall responses in the lungs during secondary heterologous infection are affected by the initial infectious dose. Memory mice [6 days p.i.; infected initially with. 1 or. 1 PFU of X-1 (HN)] were reinfected with 1 PFU of PR8 (H1N1), and antigen-specific memory CD8 T cell responses were examined on Days, 4, and 9 postsecondary infection. Cells were stimulated with NP or PA viral peptides and stained as described in Materials and Methods. Flow cytometric analyses identified NP (A, C, E) and PA (B, D, F) specific effector CD8 T cells producing IFN- in BAL, lungs, and MLN. Data obtained with mice from infected groups were compared and analyzed using Student s t test (P.). Results represent two independent experiments performed with three to four mice/experiment. Nb of virus-specific effector memory CD8 T cells (CD8 CD44 hi CD6L lo IFN-γ ) C E Lungs 4 9 MLN D F. 6. x 1.8 x 1. x 1. x 1. x x x 1 1. x x Nb of virus-specific effector memory CD8 T cells (CD8 CD44 hi CD6L lo IFN-γ ) x x x 1 4. x Lungs 4 9 MLN X-1,. x 1 PFU PR8, 1 PFU X-1,. x 1 PFU PR8, 1 PFU massive recruitment has also been associated to lung injury and increased morbidity [68]. Innate immune cells produce inflammatory cytokines, ROS and RNS, in response to pathogens, through specific enzymes. When produced at high concentrations, these ROS and RNS can have damaging effects on tissues at the site of infection [19]. Our results demonstrated that a high dose of virus leads to significant inos expression in the lungs of mice early in the course of infection. This correlates with accumulation of neutrophils, macrophages, and DCs, as well as with increased morbidity. Furthermore, oxidative stress induces antioxidant gene expression (e.g., NQO1 and HO-1), which can be used as a proxy to evaluate the oxidative stress response. We showed that NQO1 and HO-1 were increased at the same time-points, suggesting that significant oxidative stress was present in the lungs of high-dose-infected mice. As expected, mice that had received a low infectious dose of virus showed little morbidity, lower lung viral titers, a modest increase in innate immune cells (neutrophils, NK cells, macrophages, and DCs), and low oxidative and nitrosative stress responses. This suggests that ROS and RNS might contribute to morbidity in response to high-dose influenza. We repeated these experiments using the H1N1 PR8 strain to verify whether the effects of the infectious dose were specific to HN (X-1) infection. Interestingly, similar observations were made regarding the innate immune response in mice infected with two different doses of PR8 virus (. PFU vs. PFU). Thus, the effects of the initial infectious dose on the innate immune cell response might represent a common feature of influenza infection among different strains. Of note, we demonstrated that the influenza virus showed a significantly greater amplification rate (1-fold) in mice that had been infected with low-dose X-1 virus compared with those that had been infected with a high dose of virus. This suggests that viral amplification is limited in mice that received a high number of viral particles during initial in- 116 Journal of Leukocyte Biology Volume 9, July 1

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