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 low‐dose primary infection as a result of the generation of a smaller tissue‐resident CD8 + T EM cell pool.

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 H5N1 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.

Under certain circumstances, the hostˈs immune response can be inappropriate and contribute to the pathogenicity of influenza infection [ 38 ]. For example, high pathogenicity influenza infections have been shown to cause severe illness and elevated mortality rates in several hosts [ 39 – 42 ]. 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 H5N1 and 1918 H1N1 infections have revealed that the excessive inflammatory response leads to ALI, severe respiratory distress syndrome, and multiorgan failure [ 43 , 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 [ 45 ]. However, despite the fact that the adaptive immune responses to influenza have been studied extensively in the mouse [ 22 , 25 , 28 , 32 , 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.

Several viral factors contribute to influenza virulence in the host. These include sialic acid receptor use (preferentially α2,6‐linked for human viruses and α2,3‐linked for avian viruses [ 1 ]), the influenza HA glycoprotein itself [ 2 , 3 ], PB1‐F2, PB2 [ 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 [ 10 , 11 ]. Human influenza viruses are more restricted to the respiratory system, where the relevant enzymes are expressed [ 12 , 13 ]. 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‐α and IFN‐β), IL‐1β, IL‐6, TNF‐α, CCL3, CCL4, CXCL10, and CCL5 [ 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 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 [ 20 , 21 ]. In fact, the generation of a potent CD8 + T cell response is critical for the resolution of the infection [ 22 – 26 ]. 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 8–10 days p.i. [ 27 – 30 ]. 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 [ 25 , 31 ]. Upon antigen recognition, CD8 + effector T cells use several distinct effector mechanisms to eliminate invading viruses, such as the generation of antiviral mediators (IFN‐γ and TNF‐α) and direct destruction of infected cells through perforin‐, granzyme‐, TRAIL‐, and Fas ligand‐mediated mechanisms [ 27 , 32 , 33 ]. 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 ) [ 34 – 36 ]. These populations persist in the host and can be recalled to provide partial‐to‐full protection against secondary infection [ 34 , 36 , 37 ].

Sera were recovered from mice, 60 days p.i., and were decomplemented by heating at 56°C for 30 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‐31 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.

Lung homogenates (200 μl) were used for RNA extraction. RNA was isolated and purified using TRIzol, according to the manufacturerˈs protocol. RNA (1 μg) 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 6000 real‐time PCR system (Corbett, Qiagen). qPCR reactions were completed as follows: 5 min at 95°C, followed by 40 cycles—25 s at 95°C, 25 s at 57.8°C, and 30 s at 72°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 2 −ΔΔcomparative threshold method [ 51 ].

Assays to determine lung viral titers were performed essentially as described previously [ 50 ]. Two days prior to infection, MDCK cells were seeded in 24‐well plates and cultured to confluence. Cells were washed twice with PBS (Wisent) and infected with 300 μl dilutions of lung homogenates in iEMEM for 1 h. Cells were washed once, and medium was replaced with sterile Avicel 1.8%, containing 1 μg/ml of TPCK‐treated trypsin (Avicel 3.6%, diluted 1:1 with 2× iEMEM). After 48 h at 37°C 5% CO 2 , cells were washed and fixed with Carnoy fixative (methanol:acetic acid, 3:1) for 20 min at 4°C. Viral plaques were revealed by staining with 1% crystal violet solution in 20% methanol/water, 5–10 min. Numbers of viral plaques were counted, and lung viral titers were calculated based on the dilution factor.

Spleens and MLNs were homogenized in Dounce homogenizers in iEMEM. Homogenized samples were filtered through a 90‐μm nylon mesh, centrifuged, and resuspended in iEMEM. Splenocyte suspensions (1 mL) were depleted of RBC using 3 mL buffered ammonium chloride solution (Geyˈs solution) for 2 min. This reaction was stopped by adding iEMEM. The suspension was centrifuged, resuspended in 8 mL iEMEM, and filtered on 70‐μm nylon mesh. Lung homogenates were obtained by gently rubbing organs over a 200‐gauge wire mesh and filtered through a 90‐μm 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 1083 (Sigma‐Aldrich) after a centrifugation of 30 min at 800 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.

At indicated times p.i., mice were euthanized by injection of Avertin (720 mg/kg). The peritoneal cavity was exposed, and the spleen was removed and placed on ice in iEMEM containing 0.1% BSA instead of FBS. The trachea was cannulated, and three BALs (3×1 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.

Female C57BL/6 and BALB/c mice (18–20 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 (2.2.2‐tribromoethanol; 240 mg/kg; Sigma‐Aldrich) and infected by intranasal instillation of 30 μl PBS, containing two different infectious doses of X‐31 virus (H3N2; 2.52×10 2 or 2.52×10 5 PFU/mouse) or PR8 virus (H1N1; 0.5 or 50 PFU/mouse). Throughout the infection time‐course, mouse weight was monitored to assess morbidity. A weight loss of >30% 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‐31 virus, mice were anesthetized and reinfected by intranasal instillation with 30 μl PR8 virus (H1N1; 10 3 PFU), 60 days after primary infection.

TPCK‐treated trypsin from bovine pancreas and crystal violet was purchased from Sigma‐Aldrich (St. Louis, MO, USA). Avicel RC‐591 (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 366–374 (ASNENMETM) and PA 224–233 (SSLENFRAYV) were used for experiments with C57BL/6 mice and the NP 147–155 (TYQRTRALV), for experiments 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).

Reduced protective immunity against lethal secondary infection is conferred by low‐dose primary infection. Memory mice, infected initially with the two initial infectious doses during primary X‐31 infection (2.52×10 2 PFU, gray line; 2.52×10 5 PFU, black line), were reinfected with a lethal dose of PR8 virus (H1N1; 10 3 PFU), 60 days after primary infection. (A) Weight loss was determined at indicated time‐points after secondary infection. Results are presented as percentage of initial weight. For comparison purposes, results obtained with nonimmune mice, infected only with the lethal dose of the PR8 virus, are presented (black dotted line). Data were analyzed using Studentˈs t tests. Asterisks and symbols indicate statistical significance of the data between the two groups of mice infected initially with X‐31. Daggers indicate statistical significance of the data between nonimmunized mice and mice immunized with 2.52 × 10 2 PFU of X‐31; †† P < 0.01. (B) Lung viral titers were also determined at indicated time‐points after secondary infection. Data represent two independent experiments performed with three to four mice/experiment at each postsecondary infection time‐point.

The persistence of tissue CD8 + T EM cells is correlated with significant immune protection against heterologous reinfection [ 36 , 59 , 60 ]. 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 3–5 p.i., our results demonstrated that mice, infected initially with high‐dose X‐31 during the primary infection, showed significantly less morbidity during lethal heterologous reinfection compared with nonimmune mice and mice infected previously with low‐dose X‐31 ( Fig. 6A ). None of the immune mice from either group died following lethal heterologous reinfection; however, mice immunized with low‐dose X‐31 lost a significant amount of weight (≤25%), up to Day 6 p.i., tracking that of 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‐31 compared with mice of the low dose group ( Fig. 6B ).

Recall responses in the lungs during secondary heterologous infection are affected by the initial infectious dose. Memory mice [60 days p.i.; infected initially with 2.52×10 2 or 2.52×10 5 PFU of X‐31 (H3N2)] were reinfected with 10 3 PFU of PR8 (H1N1), and antigen‐specific memory CD8 + T cell responses were examined on Days 0, 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‐γ in BAL, lungs, and MLN. Data obtained with mice from infected groups were compared and analyzed using Studentˈs t test (* P <0.05). Results represent two independent experiments performed with three to four mice/experiment.

As we found that the infectious dose affected the generation of CD8 + T CM and T EM cell populations in X‐31‐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. 5 ). Sixty days after X‐31 infection, memory mice were reinfected with 10 3 PFU of PR8, and influenza‐specific memory CD8 + CD44 hi CD62L lo IFN‐γ + T cell responses were evaluated on Days 0, 4, and 9 postsecondary infection, after stimulation with appropriate peptides. Our results demonstrated that prior to reinfection (Day 0), the high initial infectious dose of X‐31 tended to generate a larger population of airway‐ and lung‐resident, virus‐specific CD8 + T CM cells (for NP and PA epitopes; Fig. 5A –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‐31 influenza ( Fig. 5A –D).

During the primary adaptive immune response to influenza, a population of memory CD8 + T cells is established [ 34 , 36 ]. Two major subsets of memory cells can be distinguished: T CM (CD8 + CD44 hi CD62L hi ), which reside in secondary lymphoid tissues, and T EM (CD8 + CD44 hi CD62L 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 5.1‐fold‐greater numbers, respectively, in the airways and lungs compared with those that received only low‐dose infection ( Fig. 4B ). Furthermore, a 4.3‐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 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, 60 days p.i. (X‐31, 2.52×10 2 or 2.52×10 5 PFU). (B) CD8 + T EM cells (CD8 + CD44 hi CD62L lo ) were identified in BAL and lungs, 60 days p.i., whereas (C) CD8 + T CM cells (CD8 + CD44 hi CD62L hi ) were identified in MLN by flow cytometry. Results obtained with mice from infected groups were compared and analyzed using Studentˈs t test (** P <0.01; *** P <0.001). Data obtained from uninfected mice show the basal level of these cells. Results represent two independent experiments performed with three mice/experiment.

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 [ 58 ]. To evaluate whether the initial infectious dose affects the generation of functional antibodies, antibodies in the sera of previously infected mice (60 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 ).

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 C57BL/6 mice is dominated by two major epitopes, namely NP and PA. Our results with the X‐31 virus demonstrated that IFN‐γ + 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, 12.1‐fold) and lungs (NP‐specific, 10.5‐fold; PA‐specific, 6.7‐fold) of mice infected with the high dose of virus compared with low‐dose infection ( Fig. 3F and G ). Our results, using the PR8 virus, demonstrated a similar trend in the number of influenza‐specific CD8 + T cells recruited into the lungs and airways (BAL; Supplemental Fig. 3C and D ). No significant differences in the numbers of effector CD8 + T cells were found in the MLN of mice infected with X‐31 or PR8 ( Fig. 3E –G and Supplemental Fig. 3B–D ). Similar results were obtained when BALB/c mice were infected with the two doses of X‐31 virus ( Supplemental Fig. 4B and C ).

We next examined the impact of the initial infectious dose on the recruitment of activated effector CD8 + T cells (CD8 + CD44 hi CD62L lo ) T cells into lungs and airways of mice at Day 10 p.i. Flow cytometry analyses revealed a significantly greater recruitment of total effector CD8 + T cells into the airways (BAL; 12.6‐fold) and lungs (7.8‐fold) of mice infected with high‐dose X‐31 compared with mice that received low‐dose infection ( Fig. 3E ). 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 (50 PFU) versus low‐dose (0.5 PFU) of PR8 virus (BAL, 16.1‐fold; lungs, 6.1‐fold; Supplemental Fig. 3B ). Also, this was not dependent on host genetic background, as similar results were obtained in BALB/c mice infected with X‐31 ( Supplemental Fig. 4B ).

Chemotactic signals and influenza‐specific effector CD8 + T cell recruitment into lungs are influenced by the initial infectious dose. Chemokine gene expression of CCL3 (A), CCL4 (B), CCL5 (C), and CXCL10 (D) in the lungs of mice infected with the two initial infectious doses (X‐31; 2.52×10 2 or 2.52×10 5 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 10 p.i., and the numbers of total effector CD8 + T cells (CD8 + CD44 hi CD62L lo ) in these compartments were determined. Cells were stimulated with the NP (F) or PA (G) peptides or left unstimulated for 6 h at 37°C. IFN‐γ production was used to identify NP‐ or PA‐specific effector CD8 + T cells (CD8 + CD44 hi CD62L lo IFN‐γ + ). Results in infected groups were compared and analyzed using Studentˈs t test (* P <0.05; ** P <0.01; *** P <0.001). 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.

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, influenza‐specific CD8 + T cells, which respond to influenza virus, depend on the expression of CCR5 and CXCR3 for their differential recruitment into the respiratory tract [ 55 – 57 ]. Therefore, we first evaluated the relative gene expression of the chemotactic signals required for CD8 + T cell recruitment (namely, CCL3, CCL4, and CCL5; CCR5 ligands and CXCL10; CXCR3 ligand). Our results showed that at 8 days p.i., only CCL4 and CCL5 were increased significantly in the lungs of mice infected with low‐dose X‐31, whereas a trend of increase for CCL3 and CXCL10 expression was observed. However, in mice infected with high‐dose X‐31, the gene expression of all the examined chemokines was increased significantly compared with low‐dose‐infected mice (CCL3, 3.4‐fold; CCL4, 7.6‐fold; CCL5, 1.6‐fold; and CXCL10, 32.6‐fold; Fig. 3A –D).

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‐31 were isolated at Day 2 p.i. The number of neutrophils (CD11c lo F4/80 − CD11b hi Gr‐1 + ), NK cells (CD3 − NK1.1 + ), macrophages (F4/80 + 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 (3.9‐fold), NK cells (2.5‐fold), macrophages (10.6‐fold), and DCs (5‐fold) was greater in mice that received the high initial infectious dose compared with the low dose of X‐31 virus ( Fig. 2H ). 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‐31 infection ( Supplemental Fig. 3A ). 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 C57BL/6 mice ( Supplemental Fig. 4A ).

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 [ 54 ]. 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 high‐dose X‐31. In fact, an early induction of iNOS mRNA was detected at 2 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. 2A ). In both groups, NQO1 expression increased significantly, 18 h (0.75 day) after infection, but the more severe infection induced a higher and sustained expression until Day 4 p.i. ( Supplemental Fig. 2B ). The expression of HO‐1 also tended to be higher at Day 2 p.i. ( P =0.21) in the lungs of the mice infected with a high dose of X‐31, whereas the low‐dose infection did not lead to any induction of this enzyme ( Supplemental Fig. 2C ).

As inflammation contributes to morbidity in animals severely infected with influenza, we determined the induction of inflammatory cytokines at Day 2 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‐α, IL‐6, and IL‐1β 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‐α, 4‐fold; IL‐6, 10‐fold; IL‐1β, 4.8‐fold; Fig. 2D –F). qPCR analyses also revealed that relative gene expression of viral PA in the lungs of mice infected with high‐dose X‐31 was 4‐fold higher than in mice infected with low‐dose virus at Day 2 p.i. ( Fig. 2G ).

The initial infectious dose modulates the early inflammatory responses and the innate immune responses to influenza. Type I IFNs IFN‐α (A) and IFN‐β (B) and type II IFN IFN‐γ (C) gene expression in the lungs of mice infected with 2.52 × 10 2 or 2.52 × 10 5 PFU doses of X‐31 was determined, 18 h p.i., by qPCR analysis using specific primers. Gene expression of TNF‐α (D), IL‐6 (E), and IL‐1β (F) and viral PA (G) was also quantified 2 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 F4/80 − CD11b hi Gr‐1 + ), NK cells (CD3 − NK1.1 + ), macrophages (F4/80 + CD11c lo CD11b hi Gr‐1 − ), and DCs (CD11c hi CD11b hi Gr‐1 + ) were identified on Day 2 p.i. (I) Early CD8 + T cell infiltration into lungs (CD3 + 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 <0.05; ** P <0.01; *** P <0.001). Nb, Number.

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 IFNRs [ 52 , 53 ]. 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‐31 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. 2A – C ). Specifically, IFN‐β induction was 2‐fold higher in mice that initially received a high dose of X‐31 compared with low‐dose infection ( Fig. 2B ). In contrast, IFN‐α and IFN‐γ induction was similar in the two groups of infected mice ( Fig. 2A and C ).

Next, we assessed lung viral titers and morbidity in mice infected initially with either dose of X‐31 over a 10‐day period. Our results showed that lung viral titers peaked at Day 2 p.i., regardless of the initial infectious dose ( Fig. 1B ). However, lung viral titers in mice, infected initially with high‐dose X‐31, reached significantly higher levels at 18 h and 48 h p.i., (2631‐ 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 2 p.i. in mice infected with high‐dose X‐31 ( 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‐31 during the early days of infection, the relative viral amplification was significantly higher (125‐fold) at the peak of viral load (Day 2 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 ).

Impact of the initial infectious dose on lung damage, viral replication, and morbidity during influenza infection. Mice were infected intranasally with a low dose (2.52×10 2 PFU; gray line) or a high dose (2.52×10 5 PFU; black line) of the X‐31 virus (H3N2). (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 10‐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 Studentˈs t test (* P <0.05; ** P <0.01; *** P <0.001). Data represent two independent experiments performed with three to four mice/experiment.

Severe influenza infections with H5N1 and 1918 pandemic H1N1 viruses can cause extensive damage to the lungs of the infected host, resulting in ALI and severe respiratory distress syndrome [ 43 , 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 C57BL/6 mice with a low dose of X‐31 (H3N2; 2.52×10 2 PFU) or a 1000‐fold higher dose (2.52×10 5 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 high‐dose X‐31 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.

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‐31 (H3N2) virus (a low dose, 2.52×102 PFU, and a high dose, 2.52×105 PFU) to infect C57BL/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 (C57BL/6 vs BALB/c) and viral strain (H3N2 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 [52, 53]. 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‐β, TNF‐α, IL‐6, and IL‐1β 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 [38, 62, 63]. 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‐31, 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, 65]. 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‐α [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 2 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 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 H3N2 (X‐31) infection. Interestingly, similar observations were made regarding the innate immune response in mice infected with two different doses of PR8 virus (0.5 PFU vs. 50 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 (125‐fold) in mice that had been infected with low‐dose X‐31 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 infection and that the associated robust host immune response contributes to limit viral spread. This is in perfect agreement with studies using mathematical models of influenza infection, which show increased viral replication when a low‐dose infection is simulated compared with high initial dose simulation [69, 70]. The significant increase in IFN‐β in mice infected with the high initial dose of influenza and its potential to contribute to the generation of the antiviral state in neighboring cells, combined to increased infiltration of innate immune cells, might, in part, explain the more limited viral amplification observed in these mice. Taken together, our results demonstrate that the initial infectious dose of influenza virus determines viral replication and pathogenicity, which is associated to viral factors and to the host immune response.

DCs play a key role in bridging the innate and adaptive immune responses. Resident and circulating DCs are recruited rapidly to the site of infection and accumulate to significant numbers to capture and transport antigens to the draining LNs, where they present these antigens to T cells [71, 72]. We showed a significantly higher recruitment of the DC population into the lungs of mice infected with the high initial influenza dose. This recruitment coincided with IFN‐β gene expression (18 h p.i.) and peak viral titers (Day 2 p.i.) and preceded a significant increase in CD8+ T cell recruitment into the airways at an early time‐point (Day 4 p.i.) in these mice. This is in agreement with a recent study, which showed that treatment of mice with IFN‐β increased the recruitment of DCs into the lungs and draining LNs of influenza‐infected mice, polarized the immune response toward type 1 immunity [73], and supports the idea that the innate immune response modulates subsequent adaptive immunity.

In this study, we demonstrated that the initial infectious dose also had a significant impact on the expression of lymphocyte chemotactic factors CCL3, CCL4, CCL5, and CXCL10 in the lungs of infected mice at later time‐points (8 days p.i.). These chemokines are essential for the recruitment of effector CD8+ T cells into the airways of infected mice via CCR5 and CXCR3, respectively, for CC and CXC chemokines, expressed at the surface of these cells [55–57, 74]. In fact, the higher infectious dose led to a significant increase in gene expression of all four chemokines studied compared with mice infected with a low dose of virus. This correlated with a significantly higher recruitment (up to 14‐fold) of total activated effector CD8+ T cells (CD8+ CD44hi CD62Llo IFN‐β+), as well as activated virus‐specific effectors (NP‐ or PA‐stimulated CD8+ CD44hi CD62Llo IFN‐γ+) into the airways and lungs of infected mice. Interestingly, morbidity in mice, infected initially with a high dose, continued to increase, although viral titers returned to similar levels in both groups of mice at Day 4 p.i. These results suggested that inflammation, a robust innate immune response, and the significant accumulation of adaptive cytokine‐producing effector CD8+ T cells in the lungs of mice, infected initially with a high dose of influenza virus, contribute significantly to the morbidity observed in these mice. This is supported by a study, which showed that TCR‐transgenic, virus‐specific cytotoxic CD8+ T cells play an important role in the pathologic manifestations of influenza with increasing doses of virus [45]. In addition, we found that although the numbers of total and antigen‐specific effector CD8+ T cells were only slightly higher in the MLN of the high infectious dose group, these numbers were comparable with those found for the low‐dose group. This suggests that as there was no obvious retention of these cells in the MLN, T cell trafficking mechanisms were not altered by the initial infectious dose. Although we did not test this specifically, taken together, these results, along with the significant recruitment of effector CD8+ T cells into the airways and lungs of mice, infected initially with a high dose of virus, suggest that a greater proliferation of effector CD8+ T cells occurred in these mice.

It is well known that the host genetic background influences the type of immune responses that is generated toward pathogens. For instance, the C57BL/6 mouse is prone to generate a Th1 response, whereas the BALB/c mouse is more prone to develop a Th2 response upon infection with the same pathogen. A good example is infection with the parasite Leishmania major, which leads to the differentiation of Th1 lymphocytes in the C57BL/6 mouse and to the generation of a Th2 lymphocyte response in the BALB/c mouse [75, 76]. Recently, some reports have described an impact of the host genetic background on influenza pathogenesis and on the inflammatory response to the virus [77, 78]. Indeed, in a recent study, variations in the intensity of the inflammatory response have been noted between different mouse strains. Interestingly, the gene expression of some cytokines, such as CCL3, keratinocyte‐derived chemokine, G‐CSF, and TNF‐α, has been shown to vary significantly in early days after infection between the C57BL/6 and BALB/c strains [77]. These studies have also demonstrated that the host genetic background affects the mortality and lung viral titers in mice. Therefore, to evaluate the effect of the infectious dose in mice with different genetic backgrounds, we performed additional experiments with BALB/c mice. Despite different genetics, our results revealed that the BALB/c and C57BL/6 mice responded similarly to the variation in the infectious dose in terms of innate and cellular adaptive immune responses. In both strains of mice, a high‐dose infection induced a much stronger recruitment of macrophages, neutrophils, NK cells, DCs, and total and specific CD8+ T cells compared with low‐dose infection. In addition to the independence on host genetic background, innate and adaptive immune responses observed with two infectious doses of PR8 (H1N1; 0.5 or 50 PFU) confirmed that these responses are also independent of the viral strain. Variations in the magnitudes of the responses between the two influenza strains could be attributed to differences in the kinetics of the immune responses. Indeed, the immune responses induced by PR8 appear to be slightly delayed compared with those induced by the X‐31 strain, which also correlates with a delayed weight loss.

After influenza virus is cleared from the lungs of infected mice, the effector CD8+ T cell response contracts, and two main populations of memory CD8+ T cells are established (T CM : CD8+ CD44hi CD62Lhi; tissue‐resident T EM : CD8+ CD44hi CD62Llo) [34–36]. We found that the initial infectious dose had a significant impact on the generation of memory CD8+ T cells of both subsets. In fact, we demonstrated that there were significantly higher numbers (up to 6‐fold) of tissue‐resident T EM in the airways and lungs and T CM (4.3‐fold) in the MLN of mice that had been infected with a high infectious dose of influenza compared with mild infection, 60 days p.i. It has been shown that tissue‐resident CD8+ T EM cells play a crucial role in controlling secondary heterologous influenza infections [59]. These cells do not proliferate but have immediate effector functions and cytolytic activity (reviewed in ref. [53]). On the other hand, circulating memory CD8+ T cells can be recruited rapidly from the circulation by inflammatory signals following heterologous secondary infection and participate in viral control by secreting antiviral cytokines within the first 4 days of reinfection [57]. Finally, CD8+ T CM cells proliferate in the LNs and secondary effector CD8+ T cells are recruited to the lungs of infected mice to amplify the memory recall response [53]. We found that during the memory phase (60 days p.i.), mice that had been infected initially with a high influenza dose had greater numbers of tissue‐resident NP and PA antigen‐specific CD8+ T EM cells in the airways and lungs compared with mice that had been infected initially with a low dose of influenza. Taken together, these results suggest that mice that initially received a high infectious dose should have enhanced protective immunity to secondary heterologous infection, as these tissue‐resident T EM cells are poised to respond to infection immediately. Interestingly, on Day 4 postsecondary heterologous H1N1 infection, our results showed that the numbers of antigen‐specific effector CD8+ T cells were similar in the two groups of mice, indicating that T CM cells could proliferate, be activated and differentiate into effectors that are recruited to the site of infection. This is supported by a study, which demonstrated that memory CD8+ T cells are recruited rapidly into the lungs through CCR5‐dependent signaling [57]. Accordingly, this population of memory cells continued to accumulate in the airways and lungs of infected animals on Day 9 postsecondary heterologous H1N1 infection; however, this was reduced in animals that had been infected initially with a high dose of influenza compared with those that had received a low dose of virus during primary infection. This is consistent with the initial protection provided by tissue‐resident memory cells in the high initial dose group. No significant differences in influenza‐specific CD8+ T EM cells were observed in the MLN in both groups of mice, suggesting that there was no defect in their generation and that T cell trafficking mechanisms were not influenced by the initial viral dose during primary infection at this point.

Accordingly, we showed that the initial infectious dose of influenza affects immune protection against secondary heterologous H1N1 infection, as a significant difference in morbidity was observed between groups of mice. In fact, mice that had been infected initially with a low dose of X‐31 (H3N2) influenza had increased morbidity significantly compared with mice that had been infected initially with a high dose of H3N2 influenza when reinfected with a lethal dose of H1N1 PR8 virus, up to 6 days postsecondary infection. The weight loss observed in mice, infected initially with a low dose of X‐31, actually tracked that of nonimmune mice during this period. Strikingly, these mice started recovering from infection on Day 7 p.i., reaching weight levels similar to those seen in mice that had been infected initially with a high dose of X‐31 during primary infection. These results are consistent with our findings that a greater number of tissue‐resident, antigen‐specific CD8+ T EM cells are found in the airways and lungs of mice infected with a high initial infectious dose of X‐31, where these cells could rapidly control infection and thus, reduce morbidity. The fact that mice from the low initial influenza dose group recovered at a later time‐point is consistent with the fact that tissue‐resident memory cells were absent but that T CM cells could be activated and differentiate into effectors which are recruited from the circulation to control secondary heterologous infection.

Although it is not possible to generalize to humans, our findings in the mouse model are highly pertinent in the context of intranasal vaccination with vaccine preparations, such as FluMist. These vaccines generate humoral and cellular immune responses to influenza and have been shown to protect against homologous and heterologous infection [58, 79, 80]. In this context of vaccination, our results demonstrated that the neutralizing antibody response was unaffected by the initial infectious dose, indicating that low‐dose vaccination would be sufficient to generate adequate antibody protection. Conversely, our results indicate that infection with a low dose of virus would provide limited protective immunity against heterologous reinfection mediated by the recruitment of effectors cells arising from CD8+ T CM cells and that this cellular protection would be delayed. On the other hand, infection with a higher dose of virus more readily generates tissue‐resident, virus‐specific effector CD8+ T cells, which react to infection immediately, thus providing significant protection that limits viral replication and morbidity. Therefore, vaccine concentration might be an important consideration for the development of cross‐protective immunity.