Nasal administration of MHV-1 dramatically reduced mortality and morbidity after heterologous virus infection. We delivered mouse hepatitis virus type 1 (MHV-1; 104 PFU) intranasally in a small volume (1 μl in each nostril) to generate nasal-only inoculation (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.99025DS1). Using this amount of inoculum, we detected viruses in the nasal lavage fluid (NLF) only on day 1 after infection (p.i.), but did not detect virus in the lungs during the course of the experiment (Supplemental Figure 1C). No virus was detected in the lungs by plaque assay or qRT-PCR. Clinically, mice with such nasal inoculum did not lose weight (Supplemental Figure 1D) or display any signs of MHV-1 pneumonia. Cell numbers (Supplemental Figure 1E) and differential in bronchoalveolar fluids (BALFs) from mice after intranasal MHV-1 were indistinguishable when compared with control mice intranasally treated with vehicle. The major cell type in BALFs from both groups was alveolar macrophages, which were defined by high autofluorescence and CD11c, SiglecF, and F40/80 expression as described previously (8). Both groups had very few T cells (CD3+), B cells (CD19+), neutrophils (Ly6G+CD11b+), or NK cells (NKp46+CD3–) cells in the BALF (data not shown). Additionally, nasal infection by MHV-1 caused significant lymphocyte expansion in the cervical lymph nodes but not in the MLNs, consistent with the presence of virus in the upper airway but not in the lungs (Supplemental Figure 1F). Since we failed to observe any signs of direct virus infection of the lungs after intranasal inoculation with 104 PFU MHV-1 in 2 μl, we used this volume/dose in all subsequent experiments (designated as “nasal-only inoculation”). We next analyzed the early immune response in the nasal-associated lymphoid tissue (NALT; Supplemental Figure 1G) and superficial and deep cervical lymph nodes within 24 hours after nasal-only inoculation. We observed that upregulated B cell expression of Ly6C (Supplemental Figure 1H) was one of the earliest events in the NALT after nasal-only MHV-1 inoculation, suggestive of B cell activation. Further, the B/T cell ratio, B cell frequency, and numbers in the NALT but not the cervical lymph nodes were significantly reduced within 12–24 hours p.i. (Supplemental Figure 1, I–L), suggesting that B cell egress from the NALT contributed to the immune response in a distant organ, the lungs.

We next examined whether nasal-only inoculation with MHV-1 could modulate the mortality and morbidity of lethal pneumonia by SARS-CoV and IAV. BALB/c mice were intranasally treated with MHV-1 (104 PFU in 2 μl). Two days later, mice were intratracheally (IT) infected with unrelated viruses, IAV or SARS-CoV, at a lethal dose. Prior nasal MHV-1 inoculation dramatically reduced the mortality rate of SARS-CoV pneumonia from 100% to 0% (Figure 1A). Morbidity, represented by weight loss, was significantly reduced (Figure 1B), whereas the kinetics of SARS-CoV clearance was significantly enhanced (>10 fold) in the lungs of mice with prior nasal MHV-1 inoculation (MHV+SARS-CoV) (Figure 1C). Histologically, mice without prior nasal MHV-1 inoculation exhibited evidence of extensive alveolar damage characterized by alveolar edema with inflammation and vascular congestion/hemorrhage, while in mice with prior nasal MHV inoculum, only minor interstitial inflammation and minor vascular congestion were observed (Figure 1, D–G). To extend these results to a non-CoV infection, we infected mice with mouse-adapted IAV (PR-8 strain, 1,160 tissue culture infectious units [TCIU] in 50 μl per mouse via IT instillation) following MHV-1 priming. Similar to SARS-CoV infection, prior nasal MHV-1 inoculation significantly reduced IAV pneumonia–induced mortality (100% to 60%) (Figure 1H), weight loss (Figure 1I), and clinical scores (Figure 1J). Lung histological examination revealed less severe alveolar wall damage in the group with prior MHV-1 inoculation (Figure 1, K–N). Taken together, these data indicate that nasal-only inoculation with MHV-1 generated robust protection against lethal pneumonia caused by heterologous viruses.

Figure 1 Nasal administration of MHV-1 dramatically reduced mortality and morbidity of lethal pneumonia by heterologous viruses. BALB/c mice were intranasally infected with MHV-1 (2 μl, 104 PFU) or vehicle. Two days later, mice were infected with SARS-CoV (104 PFU) or IAV (PR-8 strain, 1,160 TCIU) (50 μl per mouse via IT instillation). The negative controls were mice that only received vehicle. Mortality and morbidity were then monitored daily. Mice that lost >30% of their initial weight were euthanized per institutional IACUC protocols. (A) Survival rates of SARS-CoV–infected mice. P < 0.0001, SARS (n = 10) vs. MHV+SARS (n = 10) and MHV+vehicle (MHV+Veh) (n = 5). No difference was found between MHV+SARS and MHV+Veh, using both log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test. (B) Weight is expressed as percentage of original weight. **P < 0.01, among MHV-SARS (n = 10), SARS (n = 10), and MHV+Veh (n = 5) using repeated-measures ANOVA; P < 0.01, MHV-SARS vs. SARS, using LSD. (C) Viral titers at 6 days p.i. **P < 0.001, MHV-SARS (n = 5) vs. SARS (n = 5). (D–G) Lung histology: D (×10) and E (×40), representative from MHV-SARS group; F (×10) and G (×40), representative from SARS group. Hemorrhage (arrows), hyalinization (arrowheads), and extensive infiltration of inflammatory cells in both alveolar space and lung parenchyma are illustrated. (H) Survival rate. **P < 0.01, both infected groups (n = 10 for each) vs. controls (n = 6); and MHV-IAV (n = 10) vs. Veh-IAV (n = 10). (I) Weight was expressed as percentage of original weight. **P < 0.01, among MHV-IAV (n = 10), Veh-IAV (n = 10), and controls (n = 6). (J) Clinical scores on day 9 p.i. **P < 0.01, Veh-IAV vs. MHV-IAV, and both Veh-IAV and MHV-IAV vs. Veh-Veh. n = 5 for each group. (K–N) lung histology. K (10×) and L (40×), representative from MHV-IAV–infected mice; M (10×) and N (40×), representative from IAV-infected mice. Hemorrhage (arrows) and extensive infiltration of inflammatory cells (N) in both alveolar space and lung parenchyma are illustrated. Modest amounts of congestion and inflammatory infiltration in the alveolar septum was found in mice with MHV-IAV infections (thin arrow). Data are from at least 2 independent experiments. IN, intranasal; Inf Mon, inflammatory monocytes.

Nasal-only inoculation of MHV-1 remotely primes lung innate immunity by recruiting Ly6C+ IMs. To determine whether this MHV-1 protective effect was accompanied by changes in infiltrating cell composition, we analyzed mouse lungs on days 0, 1, 2, 4, and 20. While total cell numbers in the lungs did not chang after nasal-only inoculation (Figure 2A), the cellular composition was significantly altered, with decreased frequencies of both T and B cells, and increased frequency of Ly6C+ IMs (Ly6C+CD11b+CD11cintF4/80intCD3–CD19–SiglecF–Ly6G–) (Supplemental Figure 2A; Figure 2, B–D) and NK cells (Supplemental Figure 2B) at day 2 and 4 p.i. These results were further supported by nasal-only inoculation of MHV-JHM, a neurotropic coronavirus (9, 10), which also caused infiltration of Ly6C+ IMs in the lungs (3,000 PFU, 1 μl ×2 for intranasal delivery; Supplemental Figure 2, C and D). After nasal-only inoculation, MHV-JHM–infected mice developed no signs of clinical disease.

Figure 2 Nasal administration of MHV-1 remotely recruits Ly6C+ IMs into the lungs. (A) Total cell numbers in the lungs after nasal-only MHV-1 infection. n = 3–7 per group, pooled from 2 independent experiments. No differences were observed. (B and C) Ly6C+CD11b+ cell infiltration in the lungs after nasal infection. Data in B are expressed as percentage of CD45+ cells. **P < 0.01 vs. dpi 0. Gating strategy is shown in C. n = 3–12, pooled from 3 different experiments. (D) Phenotypic analysis of Ly6ChiCD11b+ cells. Blue lines represent the Ly6ChiCD11b+ cells; red lines represent negative or positive controls in each small panel as indicated. Blue-filled represents F4/80 negative control. Ly6ChiCD11b+ cells were Ly6G–CD19–CD3-SiglecF–NKp46–F4/80int. Neut, neutrophils; T, T cells; B, B cells; NK, NK cells; aM, alveolar macrophages. (E–G) Localization of Ly6C+ IMs in the lungs. BALB/c mice were intranasally infected with MHV-1 (104 PFU in 2 μl MEM, 1 μl/nostril) or vehicle, and were then sacrificed at 2 dpi. The left lungs were clamped, and the right pulmonary vessels were then exclusively perfused via the right ventricle (E). The frequencies of Ly6C+ IMs in both left and right lungs were then analyzed and expressed as percentage of CD45+ singlet cells (F and G). **P < 0.01 vs. controls. n = 3 per group.

Ly6C+ IMs that accumulated after nasal-only inoculation could be present in the lung parenchyma, alveolar spaces, or blood vessels. To distinguish these possibilities, we performed unilateral perfusion of the pulmonary vessels and examined whether such intervention changed the frequency of these cells in comparison to the non-perfused side in the same animals (Figure 2E). As shown in Figure 2, F and G, the frequency of these cells was not changed by perfusion of the pulmonary circulation, indicating that they were not circulating in pulmonary blood vessels, although they could still be adherent to vessel walls (11) or in the lymphatic vessels. The lack of change in cell number (Supplemental Figure 1E) or differential in BALFs after nasal-only inoculation suggests that the Ly6C+ IMs are situated primarily in the lung parenchyma but not in the pulmonary vessels or alveolar spaces.

Nasal-only inoculation–induced infiltration of Ly6C+ inflammatory monocytes into the lungs is IFN-I independent. Previous studies have shown that the recruitment of Ly6C+ IMs to sites of virus infection in the lung is IFN-I dependent (12–14) . In order to determine whether IM infiltration into the lungs after nasal-only inoculation was also IFN-I dependent, we infected mice lacking IFN-I receptors (BALB/c IFNAR–/–) and BALB/c controls with MHV-1 intranasally (2 μl, 104 PFU) or IT (50 μl 104 PFU), or with vehicle. Lungs were then analyzed on day 2 p.i. (Figure 3). As expected, direct lung infection with 104 PFU MHV-1 significantly increased the accumulation of Ly6C+ IMs in the lungs of WT mice but not IFNAR–/– mice. In marked contrast, in mice intranasally primed with MHV-1, Ly6C+ IMs equivalently infiltrated the lungs of IFNAR–/– and WT mice. Recent studies have demonstrated an important role for the vagus nerve in the regulation of excessive immune responses in the GI tract and lungs (15–17). Next, to assess whether the vagus nerve has a similar role after nasal-only MHV-1 inoculation, we performed unilateral cervical vagotomy (18). The contralateral side was sham-treated without vagotomy. Mice were then intranasally treated with MHV-1 (2 μl, 104 PFU) 14 days later. As shown in Supplemental Figure 3, there was nearly identical Ly6C+ IM infiltration in the lungs in vagotomized and sham-treated sides of the same animals. Taken together, these data indicate that Ly6C+ IM recruitment in the lungs after nasal MHV-1 priming is IFN-I– and vagus nerve–independent, and thus mechanistically different from that caused by direct lung infection.

Figure 3 Ly6C+ IM recruitment by nasal MHV-1 infection is not IFN-I dependent. IFNAR–/– and BALB/c mice were treated with MHV-1 intranasally (IN, 2 μl, 104 PFU) or IT (50 μl, 104 PFU) or vehicle. The mice were sacrificed at 2 days p.i. The frequency and numbers of Ly6C+ IMs in the lungs were then determined. (A) Flow cytometric plots of representative mice from each group. WT denotes BALB/c mice. (B and C) Frequency and numbers of Ly6C+ IMs in the lungs. **P < 0.01 vs. IFNAR–/– in MHV-1 IT group. n = 4–5 per group.

Ly6C+ IMs recruited by nasal-only inoculation are less activated/mature than those recruited by IT infection. We next compared the expression levels of activation/maturation markers including CD11c, MHC class II (MHCII), CCR7, CD86, CD80, and CD40 on Ly6C+ IMs recruited by nasal-only as opposed to IT MHV-1 infection. As shown in Figure 4, A–C, nasal-only and IT infection MHV-1 resulted in nearly identical frequencies and numbers of Ly6C+ IMs in the lungs on day 2 p.i. However, in mice with nasal-only inoculation, the expression levels of CD11c, MHCII, CD86, CD80, and CD40 on Ly6C+ IMs were increased to a lesser degree than after IT infection (Figure 4D), indicating the cells were less activated/mature than those recruited by direct lung infection.

Figure 4 Activation markers on Ly6C+ IMs in mice after nasal-only and IT MHV-1 infection. BALB/c mice were treated with MHV-1 intranasally (2 μl, 104 PFU) or IT (50 μl 104 PFU), or with vehicle. Their lungs were then analyzed at 2 days p.i. by flow cytometry. (A) Flow cytometric plots of representative mice from control, IN, and IT groups. (B and C) Frequencies and numbers of Ly6C+ IMs. **P < 0.01 vs. controls. No differences in either frequencies or numbers between IN and IT groups was detected. n = 6–8 per group. (D and E) Histogram and mean fluorescence intensity (MFI) of activation markers (representative of two independent experiments). Blue lines, IT; red lines, IN; green lines, control. *P < 0.05, **P < 0.01, vs. controls in each panel. n = 3 per group.

Ly6C+ IMs recruited by nasal priming can produce TNF. Previous studies have shown that secretion of TNF is one major antiviral mechanism employed by Ly6C+ IMs (13, 19, 20). To assess whether Ly6C+ IMs recruited by nasal MHV-1 priming produced TNF, we harvested lung-derived cells on day 2 p.i. and treated them with LPS (1 ng/μl) or vehicle (RP10) for 6 hours directly ex vivo, followed by intracellular staining for TNF. In the absence of LPS treatment, Ly6C+ IMs from both noninfected and MHV-1–primed mice did not produce TNF, as there was no difference between isotype-stained cells and TNF antibody–treated cells in the 2 groups (Figure 5A). After LPS stimulation, mice with nasal-only inoculation exhibited significantly more TNF-secreting Ly6C+ IMs in the lungs, in comparison to noninfected controls (Figure 5, A–C). There was no difference in the amount of TNF expressed per cells, however, based on measurements of MFI (data not shown). These data showing increased LPS-induced TNF production by Ly6C+ IMs recruited to the lungs following nasal-only inoculation with MHV-1 suggest that nasal exposure to virus-primed immune cells in the lung generates a more robust innate immune response to pathogens.

Figure 5 Intracellular expression of TNF by IMs. BALB/c mice were treated with MHV-1 intranasally (2 μl, 104 PFU, infected, n = 6 per group) or vehicle (noninfected, n = 3 per group). Their lungs were harvested to prepare single-cell suspension at 2 dpi. The cells were then treated with LPS (1 ng/μl) or vehicle (RP10) in the presence of GolgiPlug for 6 hours at 37°C. Intracellular staining for TNF was then performed. Isotype was used for all samples for optimal gating. (A) Representative flow cytometric plots from each group. (B and C) Frequency and number of TNF-producing Ly6C+ IMs. **P < 0.01. Representative of 2 independent experiments.

Increased TNF and INF-β in mice with nasal-only inoculation after SARS-CoV infection. To assess whether prior nasal-only MHV-1 inoculation generated a more robust innate immune response after SARS-CoV challenge, were infected mice intranasally with MHV-1 and challenged them with SARS-CoV at 2 days p.i. As shown in Figure 6, A and B, both TNF and IFN-β levels were dramatically increased at 24 hours after SARS-CoV infection in the MHV-1–primed mice, further supporting the notion that nasal-only inoculation enhanced the lung innate immune response after lethal virus infection.

Figure 6 INF-β and TNF expression in vivo 24 hours after SARS-CoV infection. BALB/c mice were intranasally infected with MHV-1, followed by IT infection with SARS-CoV. (A) IFN-β and (B) TNF levels 24 hours after SARS-CoV infection were determined using qRT-PCR. *P < 0.05, MHV+SARS vs. SARS. n = 4 per group.

Migration of lung Ly6C+ IMs in mice after nasal-only inoculation. Since infiltrating Ly6C+ IMs may function as APCs to orchestrate adaptive immunity (21–24), we next examined whether intranasal MHV-1 priming also augmented APC numbers and function. After nasal-only inoculation, not only was the number of Ly6C+ IMs in the lungs increased vs. controls, but the fraction expressing CCR7 also increased (Figure 7, A–C). Since CCR7 expression is required for APC migration to draining lymph nodes, we next instilled OVA-FITC IT into the lungs of mice and analyzed mediastinal lymph nodes (MLNs) 18 hours later for FITC+ cell migration. Mice with nasal infection but not controls exhibited more FITC+ Ly6C+CD11b+ IMs in MLNs (Figure 7, D–F), demonstrating that Ly6C+ IMs recruited by nasal MHV-1 inoculation were primed for both antigen uptake and migration to the MLNs.

Figure 7 Migration of Ly6C+ IMs. (A–C) CCR7 expression on Ly6C+ IMs. (A) Flow cytometric plots showing increased CCR7+Ly6+ IM levels in mice after nasal-only inoculation. (B and C) Frequencies of CCR7+Ly6+ IMs in the lungs of mice with and without nasal infection. **P < 0.01; *P < 0.05. n = 5–6 per group. (D) Migration of Ly6+ IMs into the MLNs. BALB/c mice were intranasally infected with MHV-1 (2 μl, 104 PFU) or vehicle. Two days later, FITC-OVA was instilled IT. MLNs were then collected to evaluate cell migration. Data are representative of 2 independent experiments. (D) Flow cytometric plots showing increased frequency of FITC-OVA+Ly6C+ IMs in the MLNs. (E and F) Frequencies and numbers of FITC-OVA+Ly6C+ IMs in the MLNs collected from mice with and without intranasal infection. **P < 0.01 vs. controls. n = 3 per group.

Enhanced adaptive immune response against heterologous virus challenge in the lungs after nasal infection. To assess whether enhanced Ly6C+ CD11b+ IM migration to the MLN resulted in a stronger T cell response in the lungs of MHV-1–primed mice, were treated mice intranasally with MHV-1 2 days prior to challenge with a lethal dose of SARS-CoV. The SARS-CoV–specific T cell response was then investigated using an intracellular cytokine staining assay after stimulation with virus-specific peptides (N353 for CD4+ T cells; S366 for CD8+ T cells) (25). As shown in Figure 8, prior nasal inoculation of MHV-1 significantly enhanced the SARS-CoV–specific CD4+ and CD8+ cell responses in the lungs, suggesting that enhancement of the T cell response resulting from nasal priming by MHV-1 is one possible mechanism underlying the observed protection against viral pneumonia.