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1insight jci org https doi org 10 1172 jci insight 99025 RESEARCH ARTICLE Authorship note XH and RV contributed equally to this work Conflict of interest The authors have declared that no conflict of interest exists Submitted December 4 2017 Accepted April 19 2018 Published June 7 2018 Reference information JCI Insight 2018 3 11 e99025 https doi org 10 1172 jci insight 99025 Nasal priming by a murine coronavirus provides protective immunity against lethal heterologous virus pneumonia Xiaoyang Hua 1 Rahul Vijay 2 3 Rudragouda Channappanavar 3 Jeremiah Athmer 3 David K Meyerholz 4 Nitin Pagedar 1 Stephen Tilley 5 and Stanley Perlman 2 3 1 Department of Otolaryngology Head and Neck Surgery 2 Interdisciplinary Program in Immunology 3 Department of Microbiology and Immunology and 4 Department of Pathology University of Iowa Iowa City Iowa USA 5 Department of Medicine University of North Carolina at Chapel Hill Chapel Hill North Carolina USA Introduction Epidemic and pandemic lethal viral pneumonia periodically occur resulting in substantial morbidity and mortality Effective treatment against lethal viral pneumonia remains lacking A major physiological role of the human nose is to protect the relatively vulnerable lungs 1 As the first point of contact of the respi ratory tract with the environment the nose has many unique histologic and anatomic features that allow it to effectively filter humidify and warm inhaled air to minimize detrimental stimulation of the lower airway and lungs While these physical type protective mechanisms are well recognized and studied little is known about whether the nose can prime immunity in the lungs and thereby protect them The human nose is an important branch of the mucosal immune system Mucosal surfaces that cover the gastrointestinal GI respiratory urinary and genital tracts function as immunologic sensors to detect dangerous signals including pathogens 2 3 Recent studies have shown that activation of the GI mucosal immune system not only contributes to the local immune response but also regulates immunity and function of distant organs including the lungs and the brain 4 6 For instance interaction between the GI mucosa and microbiota can alter both T cell and B cell responses to influenza A virus IAV infections 7 Similar to the GI system the human nose is covered by type I mucosa that comprises a single layer of epithelial cells abundant antigen presenting cells APCs goblet cells and specialized mucosal lymphoid organs Wald eyer s ring The human nose and upper airway effectively capture the majority of common immunogenic particles and droplets that contain pathogens in inhaled air While these foreign antigens bacteria and virus es can activate the local mucosal immune system to cause sino nasal inflammatory diseases little is known about the immunologic effect on tissues distal to the nose and upper airway especially the lungs In order to examine such effects we intranasally infected BALB c mice with a pneumotropic murine coronavirus CoV delivered in a very small volume 1 l in each nostril 2 of normal inoc ulate volume to avoid aspiration We failed to recover any virus in the lungs during the course of the The nasal mucosa is an important component of mucosal immunity Immunogenic particles in inspired air are known to activate the local nasal mucosal immune system and can lead to sinonasal inflammation however little is known about the effect of this activation on the lung immune environment Here we showed that nasal inoculation of murine coronavirus CoV in the absence of direct lung infection primes the lung immune environment by recruiting activated monocytes Ly6C inflammatory monocytes and NK cells into the lungs Unlike infiltration of these cells into directly infected lungs a process that requires type I IFN signaling nasally induced infiltration of Ly6C inflammatory monocytes into the lungs is IFN I independent These activated macrophages ingested antigen and migrated to pulmonary lymph nodes and enhanced both innate and adaptive immunity after heterologous virus infection Clinically such nasal only inoculation of MHV 1 failed to cause pneumonia but significantly reduced mortality and morbidity of lethal pneumonia caused by severe acute respiratory syndrome CoV SARS CoV or influenza A virus Together the data indicate that the nose and upper airway remotely prime the lung immunity to protect the lungs from direct viral infections 2insight jci org https doi org 10 1172 jci insight 99025 RESEARCH ARTICLE experiment using either real time quantitative RT PCR qRT PCR or virus titration and lung disease was absent Strikingly such limited local nasal infection significantly reduced the mortality rates after challenge with 2 heterologous pathogenic respiratory viruses SARS CoV from 100 to 0 and IAV from 100 to 60 and reduced clinical disease We observed a significant increase in numbers of Ly6C inflammatory monocytes IMs in the lungs 2 days after nasal infection Moreover Ly6C IM recruited by low volume nasal infection were less activated and proinflammatory compared with those detected after direct lung infection Our results suggest that these monocytes dramatically enhanced the innate and virus specific CD4 and CD8 T cell responses after heterologous virus challenge These findings indicate that activating the nasal innate immune response may be very useful for the prevention and treatment of lethal viral pneumonia Results Nasal administration of MHV 1 dramatically reduced mortality and morbidity after heterologous virus infection We delivered mouse hepatitis virus type 1 MHV 1 10 4 PFU intranasally in a small volume 1 l in each nos tril 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 differ ential 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 inocu lation with 10 4 PFU MHV 1 in 2 l 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 10 4 PFU in 2 l 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 Fig ure 1C Histologically mice without prior nasal MHV 1 inoculation exhibited evidence of extensive alveolar damage characterized by alveolar edema with inflammation and vascular congestion hemor rhage 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 l 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 3insight jci org https doi org 10 1172 jci insight 99025 RESEARCH ARTICLE 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 l 10 4 PFU or vehicle Two days later mice were infected with SARS CoV 10 4 PFU or IAV PR 8 strain 1 160 TCIU 50 l 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 infiltra tion 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 4insight jci org https doi org 10 1172 jci insight 99025 RESEARCH ARTICLE 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 CD11c int F4 80 int CD3 CD19 SiglecF Ly6G 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 exper iments D Phenotypic analysis of Ly6C hi CD11b cells Blue lines represent the Ly6C hi CD11b cells red lines represent negative or positive controls in each small panel as indicated Blue filled represents F4 80 negative control Ly6C hi CD11b cells were Ly6G CD19 CD3 SiglecF NKp46 F4 80 int 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 10 4 PFU in 2 l MEM 1 l 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 percent age of CD45 singlet cells F and G P 0 01 vs controls n 3 per group 5insight jci org https doi org 10 1172 jci insight 99025 RESEARCH ARTICLE 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 l 2 for intranasal delivery Supplemental Fig ure 2 C and D After nasal only inoculation MHV JHM infected mice developed no signs of clinical disease 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 paren chyma but not in the pulmonary vessels or alveolar spaces Nasal only inoculation induced infiltration of Ly6C inflammatory monocytes into the lungs is IFN I indepen dent 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 l 10 4 PFU or IT 50 l 10 4 PFU or with vehicle Lungs were then analyzed on day 2 p i Figure 3 As expected direct lung infection with 10 4 PFU MHV 1 sig nificantly 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 reg ulation 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 l 10 4 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 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 Figure 3 Ly6C IM recruitment by nasal MHV 1 infec tion is not IFN I dependent IFNAR nullnull and BALB c mice were treated with MHV 1 intranasally IN 2 l 10 4 PFU or IT 50 l 10 4 PFU or vehicle The mice were sacrificed at 2 days p i The frequency and num bers 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 Fre quency and numbers of Ly6C IMs in the lungs P 0 01 vs IFNAR nullnull in MHV 1 IT group n 4 5 per group 6insight jci org https doi org 10 1172 jci insight 99025 RESEARCH ARTICLE 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 inocula tion 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 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 l 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 inocu lation 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 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 l 10 4 PFU or IT 50 l 10 4 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 con trol P 0 05 P 0 01 vs controls in each panel n 3 per group 7insight jci org https doi org 10 1172 jci insight 99025 RESEARCH ARTICLE that nasal exposure to virus primed immune cells in the lung generates a more robust innate immune response to pathogens Increased TNF and INF in mice with nasal on ly 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 w