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. Author manuscript; available in PMC: 2024 Feb 1.
Published in final edited form as: J Immunol. 2023 Feb 1;210(3):348–355. doi: 10.4049/jimmunol.2200604

Low dose lipopolysaccharide protects from lethal paramyxovirus infection in a macrophage and TLR4 dependent process

Jenny Resiliac *,†, Michelle Rohlfing †, Jennifer Santoro †, Syed-Rehan A Hussain †, Mitchell H Grayson †,‡
PMCID: PMC9851983  NIHMSID: NIHMS1851775  PMID: 36480273

Abstract

Respiratory diseases are a major public health burden and leading causes of death and disability in the world. Understanding anti-viral immune responses is crucial to alleviate morbidity and mortality associated with these respiratory viral infections. Previous data from human and animal studies suggested that pre-existing atopy may provide some protection against severe disease from a respiratory viral infection. However, the mechanism(s) of protection is not understood. Low dose lipopolysaccharide (LPS) has been shown to drive an atopic phenotype in mice. In addition, LPS has been shown in vitro to have an antiviral effect. We examined the effect of LPS treatment on mortality to the murine parainfluenza virus, Sendai virus (SeV). Low dose LPS treatment 24 hours before inoculation with a normally lethal dose of SeV greatly reduced death. This protection was associated with a reduced viral titer and reduced inflammatory cytokine production in the airways. The administration of LPS associated with a marked increase in lung neutrophils and macrophages. Depletion of neutrophils failed to reverse the protective effect of LPS; however, depletion of macrophages reversed the protective effect of LPS. Further, we demonstrate that the protective effect of LPS depends upon type I interferon (IFN) and TLR4-MyD88 signaling. Together, these studies demonstrate pretreatment with low dose LPS provides a survival advantage against a severe respiratory viral infection through a macrophage, TLR4, and MyD88 dependent pathway.

Keywords: Animals-Rodent, Infections-Viral, Tissues-Lung, lipopolysaccharide, Sendai virus, innate immunity, Toll like receptor 4, MyD88

Introduction

Respiratory diseases are among the leading causes of mortality and disability in the world. They are an increasing public health burden globally because of the morbidity and mortality associated with severe infections in the young, elderly, and immunocompromised [1–3]. In 2013, 2.6 million deaths were ascribed to respiratory viral infections [4, 5]. The common viruses identified from these infections include influenza viruses, respiratory syncytial virus (RSV), parainfluenza viruses, human metapneumovirus (HPMV), and respiratory adenoviruses [6]. Therefore, it is most important to develop strategies that may reduce the morbidity and mortality burden accompanying these infections.

Several studies have suggested that atopic disease may provide a survival advantage with severe respiratory viral infections [7–9]. During the 2009 influenza pandemic, hospitalized asthma patients had lower mortality than those hospitalized without asthma (4% vs. 10%, respectively; p=0.04), suggesting an atopic survival advantage [10]. Similar atopic advantage has been described during the SARS-Cov2 pandemic in which asthmatic patients were reported to have better survival outcomes compared to non-atopic individuals [11, 12]. Additionally, murine models of allergic asthma reported protective effects of allergens on severe influenza viral infections[13, 14]. However, the mechanism(s) by which atopy protects from respiratory viral infection induced mortality is unknown. Interestingly, bacterial endotoxin, also known as lipopolysaccharide or LPS, has been associated with atopic disease in a dose-dependent manner based on time of exposure [15]. Lower doses of LPS have been shown to have protective effects in stimulating an immune response [16–18]. In vitro studies demonstrated that LPS can interfere with viral infections and destabilize influenza viruses by directly binding to their virions to inhibit their replication in a temperature-dependent manner [19, 20]. When added to cells prior to viral infection, LPS prevented replication of Dengue and human immunodeficiency viruses on monocytes/macrophages in vitro [21, 22]. However, little is known on the effect of prior exposure of LPS on the anti-viral immune response in vivo.

Sendai virus (SeV), also known as the murine parainfluenza virus 1, is an enveloped negative-sense single stranded RNA virus from the family Paramyxoviridae and genus Respirovirus. SeV is in the same family as human pathogens associated with asthma and atopy development such as parainfluenza virus and RSV. SeV infection has been documented to drive post-viral airway disease in a well-characterized mouse model [23–25]. Therefore, using LPS and a normally lethal dose of SeV, we determined the effects of prior exposure of LPS on the anti-viral immune response. Our study demonstrates that low dose LPS is protective against a severe respiratory viral infection, and that this protection depends upon macrophages, but not neutrophils. Further the LPS induced protection appears to be mediated through type I interferon, TLR4 and MyD88, as mice genetically unable to signal through these molecules fail to gain a survival advantage with LPS pretreatment. The TLR4-MyD88 pathway is involved in the pathogenesis of several diseases and there is increasing evidence that dampening this pathway can be protective during acute lung injury and some cancers [26–29]. However, little is known on the in vivo role of TLR4-MyD88 pathway during severe respiratory viral infection. This study is the first to our knowledge to demonstrate a protective effect of endotoxin exposure (LPS), a potent TLR4 agonist, on the murine antiviral immune response using a native murine virus and a normally lethal viral infection.

Materials and Methods

Study approvals

All animal experimental protocols were approved by the institutional animal care and use committee (IACUC) of the Abigail Wexner Research Institute at Nationwide Children’s Hospital (AWRINCH).

Mice

All mice were obtained from Jackson Laboratory (Bar Harbor, ME). C57BL/6J (WT), Toll-like receptor 4 deficient (Tlr4–/– B6. B6(Cg)-Tlr4tm1.2Karp/J, Jackson Stock 029015), Myeloid differentiation primary response 88 deficient mice (Myd88−/−; B6.129P2(SJL)-Myd88tm1.1Defr/J, Jackson Stock: 009088/Myd88 null) were purchased and bred in house. Type I IFN receptor deficient (Ifnar−/−) on C57BL/6 background were obtained from J. Sprent (The Scripps Research Institute, La Jolla, CA) [23]. Recombination activating gene deficient mice (Rag1–/–; B6.129S7-Rag1tm1Mom/J; 002216) were gifted from Drs. Ross Maltz and Masako Shimamura’s laboratories at AWRINCH. These mice were originally purchased from Jackson laboratories and bred in house.

Animal procedures

LPS administration and viral infection

6–8 weeks old C57BL/6J (wild type, WT) mice were inoculated intranasally (i.n.) with varying doses of LPS (from E. coli O55:B5, catalog # L6529, Sigma, St. Louis, MO USA) or phosphate-buffered saline (PBS) 24 hours before i.n. administration of a normally lethal dose of Sendai virus strain 52 (SeV; 2×106 pfu in 30 μL PBS) from ATCC (American Type Culture Collection, Manassas, Virginia; catalog # VR-105) or ultraviolet light inactivated SeV (UV-SeV; virus negative control). Ultraviolet light inactivation of SeV virus was performed by placing 2×106 pfu SeV in a Spectrolinker™ XL-1000 UV Crosslinker for 3 cycles of UV exposure (1200 × 100 uJ/cm2) to crosslink the viral RNA.

Bioplex assays using bronchoalveolar lavage fluid

Bronchoalveolar lavage (BAL) fluid was collected from 0.1μg LPS or PBS treated mice 6 hours post LPS/PBS, at the time of SeV inoculation or 1, 3, 5, and 7 days post inoculation (PI) SeV. In order to limit the number of mice needed for these experiments, we did not examine PBS treated mice at 6 hours after administration of PBS, but compared the LPS treated mice 6 hours post LPS with the PBS treated mice 24 hours post PBS. We utilized a Bioplex Pro-mouse cytokine 23-plex immunoassay (#m60009rdpd, Bio-Rad, California, USA) to measure BAL cytokines. Assays were performed according to the manufacturer’s instructions. Per the manufacturer, the limits of detection for the analytes were (minimum pg/ml): interleukin (IL)-1α (0.46), IL-1β (1.4), IL-6 (0.75), IL-17a (0.43), IL12p40 (5.62), tumor necrosis factor (TNF)-α (3.19), granulocyte-colony stimulating factor (G-CSF) (6.5), Regulated upon activation, Normal T cell Expressed and Secreted (RANTES/CCL5) (3.02), keratinocyte chemokine (KC/CXCL1) (1.50), Monocyte chemoattractant protein 1 (MCP-1/CCL2) (15.85), macrophage inflammatory protein 1 (MIP-1-α/CCL3) (0.21), MIP-1-β/(CCL4) (1.77), granulocyte macrophage colony stimulating-factor (GM-CSF) (2.88). Additional analytes in the assays not included in results because they were below the limit of detection or not different between PBS (controls) and LPS (experimental) groups (minimum pg/ml): IL-2 (1.17), IL-3 (0.64), IL-4 (0.43), IL-5 (0.72), IL-9 (2.34), IL-10 (3.03), IL12p70 (1.90), IL-13 (12.76), Eotaxin/CCL11 (1.06), IFN-γ (0.71). For statistical analyses, all analyte values below the limit of detection were set to a value of 75% of the minimum detection limit for the analyte.

Quantitative real-time PCR (qRT-PCR) Assay

Total RNA was isolated with Trizol (Sigma-Aldrich). Up to 2 μg of RNA was used to make cDNA. cDNA was synthesized with Maxima™ H Minus cDNA Synthesis Master Mix, with dsDNase, (cat# M1681, ThermoFisher) per manufacturer’s instructions. The StepOne Plus PCR system (Applied Biosystems) was used and qRT-PCR was performed using TaqMan fast master mix.

The primer and probe sets used for qRT-PCR experiments were:

SeV primer/probe set (SeV-1237F: 5’-GGC GGT GGT GCA ATT GAG-3’; SeV-1300R: 5’-CAT GAG CTT CTG TTT CTA GGT CGA T-3’; MGB TaqMan Probe SeV-1257T: 5’-AGC TCT AGA CAA TGC C-3’) for SeV qRT-PCR and copy number calculated based on a standard curve as previously published [30]. TaqMan expression assays (ThermoFisher Scientific, Waltham, MA) were used to assess mRNA expression of interferon-α (Ifna2) (Mm04207507_gH), Ifnb1 (Mm00439552_S1)), Ifng (Mm01168134_m1), with data normalized to Gapdh (Mm99999915_g1).

Flow cytometry

Mice were euthanized 6 hours post-LPS inoculation, 24-hours after LPS or PBS administration, and days 1 (24-hours post-viral infection), 3, 5, and 7 PI SeV. Cells were harvested from whole lungs for flow cytometry to characterize cellular infiltrates in the lungs, per our published methods [23]. Stained cells were analyzed with a FACSCalibur flow cytometer (BD) and data analyzed with FlowJo software (Tree Star, Inc). In addition to scatter, cells were identified using the following: neutrophils (GR1+), alveolar macrophage (GR1-, Siglec F+, CD11c+), interstitial macrophage (GR1−, Siglec F−, CD11b+, CD11c+, F4/80+, MHCII+) as shown in Supplemental Figure 1. Clones of antibodies used: Gr-1 (RB6–8C5); Siglec-F (R35–90); CD11c (N418); CD11b (M1/70); MHC-II (MS/114.15.2); F4/80 (BM8); Ly6G (1A8). As mentioned for the cytokine studies, in order to limit the number of mice needed for these experiments, we did not examine PBS treated mice at 6 hours after administration of PBS, but have instead compared LPS treated mice 6 hours post LPS with the PBS treated mice 24 hours post PBS.

Neutrophil Depletion

To deplete neutrophils, 100 μg of anti-Ly6G mAb (clone 1A8, BioXCell) or a control rat IgG2a (clone 2A3, BioXCell) was given via intraperitoneal injection (i.p.) immediately following LPS administration. Twenty-four hours later mice were inoculated with 2×106 pfu SeV and survival determined. Administration of anti-Ly6G reduced neutrophils in the lungs by 76.48±0.32% (mean ± SEM; compared to control IgG administration) 24 hours after antibody injection (Supplemental Figure 2A).

Macrophage Depletion

Macrophages were depleted by injecting each mouse i.p. with 2 mg of clodronate containing or empty (control) liposomes (Clodrosome + Encapsome; CLD-8901; Encapsula Nanosciences LLC). 48 hours later, mice were treated with LPS (0.1 μg of LPS i.n in 30 μL), and a second dose of 0.5 mg clodronate or empty liposomes given i.p. Twenty-four hours post LPS treatment (72 hours post the initial i.p. liposome injection), mice were infected with SeV (2 × 106 pfu). Administration of 2.5 mg clodronate reduced lung macrophages by 64.33±1.93% (mean ± SEM; compared to empty liposome administration) 6 hours after i.p. injection (Supplemental Figure 2B, C).

Statistical analyses

GraphPad Prism v.9.1.0 was used for statistical analyses. All values are expressed as mean ± SEM, unless otherwise noted. Student’s t-test (for parametric data) or Mann Whitney U (for non-parametric data) were used to assess significant differences between two means (Student’s t-test) or medians (Mann Whitney U). For all tests, p ≤ 0.05 was considered statistically significant.

Results

LPS exposure protects against viral induced mortality

To determine if LPS was protecting mice from a normally lethal SeV infection, we inoculated mice with various doses of LPS or PBS 24 hours before infection with 2×106 pfu SeV (a dose that is usually uniformly lethal to wild type (WT) C57BL/6J mice) and monitored survival. PBS treated mice all succumbed to the virus by 9 days PI SeV; however, LPS reduced mortality by at least 50% even at the highest dose in these experiments (3μg) (Figure 1A). There was an LPS dose-response relationship in the reduction of mortality with the highest rate of survival (above 80%) observed in the groups receiving 0.1 or 0.03 μg of LPS. Interestingly, these lower doses of LPS have been associated with development of atopic disease [15, 31, 32]. Additionally, we determined that pre-treating mice 24 hours prior to viral infection, but not 24 hours after viral inoculation, provided a survival advantage (Supplemental Figure 3A). For all subsequent experiments we used the 0.1 μg dose of LPS 24 hours prior to SeV infection.

Figure 1. LPS reduces mortality to SeV in a dose dependent fashion and affects viral titer early during infection.

Figure 1.

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A) Mice were inoculated with PBS or the indicated doses of LPS 24 hours before inoculation with 2×106 pfu SeV. Survival was then monitored (Mantel Cox, p=0.0001 for all groups; n≥6 per group). As a negative control, mice were treated with 3 μg LPS before inoculation with UV-inactivated SeV (UV-SeV). B) LPS (open circle) compared to PBS (filled circle) pretreated mice had significantly decreased lung viral titer at days 3 PI SeV; however, by day 5 PI SeV there was no difference in titer. Data are presented as mean ± SEM copies of SeV. Mice numbers were the following: 3 μg LPS-UV-SeV, N=2; 3 μg LPS, N=17; 1 μg LPS, N=4; 0.3 μg LPS, N=9; 0.1 μg LPS, N = 14; 0.03 μg LPS, N=13; PBS, N=14. Data polled from 3 experiments. ** p≤0.01.

LPS modestly reduces viral titer

Given reduced mortality in LPS treated SeV infected mice, and the published data that LPS can directly reduce viral replication in vitro[19, 20], we examined SeV titer. LPS treated mice had significantly reduced viral burden on day 3 PI SeV; however, by day 5 PI SeV there was no residual effect of LPS administration on viral titer (Figure 1B). These effects are rather modest and may not be impacting the infectivity of the virus nor affecting the pathology of the disease. Therefore, it did not seem to be the most likely mechanism through which LPS mediates increased survival. Hence, we examined the effect of LPS administration on the immune response to SeV.

LPS protection depends upon innate immunity

LPS is a potent immunostimulant that activates the innate immune system [33]. However, we did not know if the survival advantage conferred by LPS relied solely on the innate immune response. To test the importance of the innate immune response in mediating survival, we used Rag1 deficient mice (Rag1–/–) and determined the effect of LPS exposure on survival from SeV (using a lower dose of SeV, given the severe immunodeficiency in Rag1–/– mice) [34]. Rag1–/– mice exposed to LPS had significantly delayed death compared to PBS treated Rag1–/– mice, with death delayed by ~4 days (Supplemental Figure 3B). Since survival from SeV requires a CD8 T cell response, it is not surprising that all Rag1−/− mice succumbed to the virus [35–37]. However, this delay in death suggested to us that LPS mediated its protection through an innate immune component.

Effect of LPS on lung cellular infiltrate and airway cytokines

Based upon the requirement for an innate immune response, we next investigated the innate immune cellular infiltrate induced by LPS. Lungs from WT mice were harvested at various time points after LPS/PBS and/or SeV administration and cellular infiltrate examined by flow cytometry. Among the various immune cell types identified, neutrophils and macrophages (both alveolar, aMOs, and interstitial macrophages, iMOs) were significantly increased in lungs of LPS treated mice by frequency and cell numbers compared to controls (Figure 2). Although not surprising, as this increase of neutrophils and macrophages with LPS is well documented [38–41], it is interesting that neutrophil levels returned to baseline at the time of viral infection, while both aMOs and iMOs remained increased.

Figure 2. LPS induces robust lung recruitment of neutrophils and macrophages.

Figure 2.

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Frequency and cell counts of lung neutrophils (PMN, GR1+ cells, top panels), alveolar macrophages (aMOs, middle panels), and interstitial macrophages (iMOs, bottom panels) after administration of 0.1 μg LPS (open circle) or PBS (filled circle) i.n. at the indicated days PI SeV (LPS/PBS given 24 hours before 2×106 pfu SeV). Note day −0.75 PI SeV is 6 hours post LPS, while day 0 PI SeV is 24 hours post LPS; in both cases, these mice (day −0.75 and 0) did not receive SeV. N=10 per group at 6 hours, 24 hours, and day 1 PI SeV; N=14 at days 3, 5, and 7 PI SeV. Data pooled from 3 experiments. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Utilizing a 23-plex assay, we next assessed the effect of LPS administration on bronchoalveolar lavage (BAL) cytokines. As expected LPS induced a strong inflammatory response with increased expression of pro-neutrophil cytokines/chemokines, G-CSF, KC/CXCL1, IL-1α, IL-1β, IL-6, IL-17a, and TNF-α (Figure 3A). Additionally, LPS treatment led to increased levels of macrophage associated cytokines/chemokines such as MCP-1/CCL2, MIP-1-α/CCL3, MIP-1-β/CCL4, RANTES/CCL5, GM-CSF, and IL12p40, and IL-12p70 (Figure 3A). Interestingly, the robust inflammatory response induced by LPS was short-lived, with most cytokine levels returning to baseline by the time of viral infection – except for G-CSF and IL-12p40, which remained significantly increased one day into the viral infection. Importantly, these cytokines and chemokines are associated with recruitment and activation of neutrophils [42] and macrophages (Figure 3A) [43]. We also investigated interferons (IFN)- α, β, and γ message (mRNA expression) by RT-PCR and found them also elevated in LPS treated mice (Figure 3B).

Figure 3. Impact of LPS treatment on airway cytokines/chemokines.

Figure 3.

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A) BAL cytokines/chemokines were measured from mice treated as in Fig 2 using a multiplex array. Open circles represent LPS treated mice, while filled circles are PBS treated. B) IFN α, β, and γ mRNA expression was measured in lungs of mice treated as in (A). *p≤0.05, **p≤0.01, ***p≤0.001,****p≤0.0001; N≥6 for each timepoint for (A) and N≥4 for (B). Data pooled from 3 experiments.

Macrophages, but not neutrophils, are required for LPS mediated survival

Given the requirement for an innate immune component in providing the LPS mediated increased survival, and the increased neutrophils in LPS treated mice, we next investigated whether neutrophils were required for LPS mediated survival. Using anti-Ly6G antibodies, we depleted neutrophils at the time of LPS administration and examined the effect on survival from SeV infection. As shown in Figure 4A, depletion of neutrophils did not impair LPS mediated increased survival (i.e., no difference in survival between the anti-Ly6G and control IgG treated mice). Therefore, neutrophils do not appear to be required for the survival protection seen in LPS treated mice.

Figure 4. Macrophages, not neutrophils, and type I IFN are required for LPS mediated survival.

Figure 4.

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A) Mice treated as in Fig 2, but given 100 μg anti-Ly6G or control IgG i.p. at the same time as LPS exposure. One day later, mice were inoculated with 2×106 pfu SeV i.n. and survival monitored. N = 14 mice in the LPS+anti-Ly6G group; n=12 in LPS+IgG control group; n=2 for PBS group. Data pooled from 2 experiments. B) 2 mg of clodronate containing or empty (control) liposomes were given i.p. and 48 hours later, mice received 0.1 μg LPS and a second i.p. dose of 0.5 mg clodronate or empty liposomes. Twenty-four hours after receiving LPS, 2×106 pfu SeV was administered, and survival determined. For comparison, mice that did not receive any liposomes, but were given LPS and SeV are also included (black solid line). Mantel Cox statistical comparisons are shown on the graph. Survival was not significantly different between mice that received LPS and no other treatment and those that received LPS and empty liposomes. N=7, for treatment with clodronate and LPS; n=8, for treatment with empty liposome and LPS; n=4 for LPS with SeV only group. **p≤0.01, ****p≤0.0001. C) Wild type (WT) B6 or Ifnar−/− mice received 0.1 μg LPS or PBS i.n. and 24 h later were inoculated with 2×106 pfu SeV or UV-inactivated SeV (UV-SeV) and survival monitored. Ifnar−/−: LPS treatment group N = 13 and N=7 in PBS group. WT B6: N=8 in LPS and N=3 in PBS groups; N=2 for UV-SeV (Ifnar−/− and WT B6) control groups. Data pooled from 2 experiments. ***p≤0.001.

Macrophages have known roles in mediating innate immunity in hosts after pathogen assaults. Since macrophages were also increased in our model, as were macrophage associated cytokines, we investigated their role in facilitating the LPS mediated survival advantage. Using clodronate containing liposomes, we depleted macrophages and determined the effect on LPS induced survival from SeV. All clodronate treated mice died, while a significant number of control mice (empty liposome) survived (Figure 4B). Neutrophils were not affected by clodronate treatment (Supplemental Figure 2C). However, even the administration of liposomes led to increased mortality compared to LPS and SeV treated (no liposomes or clodronate) mice. Nonetheless, the significant difference in mortality between the clodronate and empty liposome groups strongly argues that macrophages are required for LPS mediated survival advantage to occur.

Type I IFN requirement for LPS mediated survival

LPS treatment markedly increased mRNA expression for type I IFNs (Ifna and Ifnb) before viral inoculation (Figure 3B). Since type I IFN are known anti-viral cytokines, we next examined whether type I IFN signaling was required for survival in LPS treated mice [44]. Using mice deficient in the type I IFN receptor (Ifnar−/− mice), we had previously found that Ifnar−/− mice survived a regular dose (i.e., 2×105 pfu) SeV infection, so type I IFN signaling is not required to survive a SeV infection (data not shown). Therefore, we examined the effect of LPS pretreatment on survival of Ifnar−/− mice when infected with 2×106 pfu SeV. As shown in Figure 4C, LPS provided no survival advantage in Ifnar−/− mice, indicating that type I IFN signaling is required for LPS mediated survival to SeV.

Requirement for TLR4 and MyD88 in LPS mediated protection from SeV lethality

Since TLR4 is the primary receptor for LPS, we investigated whether LPS mediated its survival via TLR4. Not surprisingly, mice deficient in TLR4 (Tlr4−/−) all succumbed to the viral insult regardless of LPS treatment (Figure 5A), but the TLR4 did not seem to be necessary for the modest effect of LPS on viral titer (Figure 5B). Notably, loss of TLR4 also prevented the LPS driven increase in lung aMOs and iMOs (Figure 5C, D), and the increase in pro-inflammatory cytokines in the BAL (Supplemental Figure 4).

Figure 5. LPS mediated survival, but not viral reduction, in a SeV infection are TLR4 dependent.

Figure 5.

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A) WT B6 or Tlr4−/− mice were treated as in Fig 2, and survival determined. WT B6: N=14 mice per group. Tlr4−/−: N=6 mice per group, ****p≤0.0001. Data pooled from 3 experiments. B) Lung SeV titers from Tlr4−/− mice treated as in (A). *p≤0.05. Tlr4−/− mice treated as in (A) and lung (C) aMOs and (D) iMOs determined by flow cytometry at the indicated times. *p≤0.05, **p≤0.01.

The adapter protein MyD88 is a co-receptor molecule for TLR4 and canonical adaptor for inflammatory pathways downstream of TLRs as well as activator protein in innate immune signaling [45]. Therefore, we hypothesized that LPS mediated protection required MyD88 signaling and examined the effect of LPS pretreatment on survival from SeV in Myd88–/– mice. Initially, we performed a dose ranging experiment to see what titer of virus Myd88−/− mice could survive. Myd88−/− mice all survived 2×104 pfu SeV infection (data not shown), so we examined the effect of LPS administration with 2×105 pfu SeV (one log higher dose than the titer that all mice survived without LPS – i.e., similar rationale as using the 2×105 pfu and 2×106 pfu doses in WT mice). As shown in Figure 6, all Myd88−/− mice succumbed to 2×105 pfu SeV regardless of whether they received LPS pretreatment or not. Based upon these data and those with the Tlr4−/− mice, we conclude that the TLR4-MyD88 complex is required for LPS mediated survival.

Figure 6. MyD88 is required for LPS mediated survival from SeV.

Figure 6.

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Myd88−/− mice treated with 0.1μg LPS (dotted line) or PBS (solid line) 24 hours before i.n. inoculation with 2×105 pfu SeV, and survival then determined. N=12 mice in LPS treated group and N=10 in PBS group. Data pooled from 2 experiments.

Discussion

The major finding of our work demonstrates that pretreatment with low dose LPS provides protection from a normally lethal respiratory viral infection in a dose dependent fashion. Additionally, while LPS is strongly associated with a neutrophilic response, in our model, neutrophils were dispensable for the protection mediated by LPS. Interestingly, macrophages were found to be required for this survival advantage. In addition, we determined that viral titer was significantly reduced early in the viral infection when mice were pretreated with LPS. Macrophages are known professional phagocytes. Therefore, it is intriguing to consider that the macrophages might play a role in reducing the viral titer, although we acknowledge this could be a direct effect of the LPS on inhibiting viral replication, as previously reported in in vitro studies, but it is important to note that in our studies the LPS was given 24 hours before the viral inoculation [21, 22]. Unlike LPS administration 24 hours before infection, LPS treatment 24 hours post viral infection was not protective from viral lethality since all these mice succumbed to the viral infection. Additionally, we have previously demonstrated that some neutrophil subsets can reduce viral titer in SeV infected mice; however, given the lack of a requirement for neutrophils in LPS mediated protection from the viral insult, we believe this is a less likely mechanism of protection [25].

Our results also show that LPS is skewing the cytokine milieu to be pro-inflammatory prior to SeV infection. Interestingly, the LPS induced inflammatory response quickly dampens by the time of viral infection while PBS treated mice had a delayed and sustained inflammatory response with increased IL-1α, IL-6, and TNF-α amongst other cytokines/chemokines post SeV infection. This reduced inflammatory response early during infection is suggestive of faster resolution of inflammation from the acute lung injury due to the infection and can be part of mechanism(s) by which LPS provides the survival advantage. Additionally, type I IFN signaling was required to survive the severe respiratory viral infection, which fit with the early IFN response seen in LPS treated mice. These observations suggest the initial early inflammatory response stimulated by LPS activates the innate immune response translating into a robust anti-viral immune response after SeV infection 24 hours later and ultimately leading to increased survival.

Macrophages were increased in the lungs of LPS treated mice and in our model appear required for the LPS mediated increased survival. We depleted macrophages with liposomes containing clodronate which has been reported to mainly affect aMOs [46, 47]. aMOs are among the first line of defense of the innate immune system and can have different phenotypes to mitigate the inflammatory response. A recent study showed repeated exposure to LPS was capable of reprogramming aMOs to have a distinct form of memory, allowing them to better recognize pathogens and enhanced phagocytic and cytotoxic activities [48]. Macrophage training is mainly characterized by reprograming of epigenetic, metabolic, and functional changes, which has been reported to occur within 3 to 5 days of repeated exposure to a stimuli [49]. This timeline might be a result of the fact that training has been shown to require T helper cell activation [50]. It is intriguing to think a similar reprogramming of aMOs was occurring in our model, since LPS was only protective when given before but not after the viral infection. However, given the single administration of LPS and the short exposure timeframe prior to the infection, this would have to be a more rapid form of training than has been previously reported. Future studies are needed to determine the impact of LPS on memory training in our model and these are beyond the scope of the current study. Nonetheless, LPS is most likely priming these macrophages leading to increased protection during the severe respiratory viral infection [51]. A caveat to our studies is that depleting macrophages with clodronate containing liposomes also reduces all phagocytes including dendritic cells, which could also be important in the reduced mortality. Future studies will be needed to determine the sufficiency of macrophages in protecting from the normally lethal dose of SeV. Although neutrophils and macrophages were the main cell types increased in our model, we acknowledge that other innate immune cell types may also be aiding the survival against SeV induced mortality.

Additionally, we have shown that TLR4-MyD88 signaling is required for the survival protection to occur. Whether the TLR4-MyD88 complex signaling is mainly required for macrophages to facilitate survival is not known and will be determined in future studies. In the absence of TLR4, the increase in neutrophils and macrophages in the lungs were abrogated regardless of LPS treatment along with the initial pro-inflammatory cytokine response. Importantly, the productions of GM-CSF, G-CSF, KC, CCL3/4/5, and IL12p40 were dependent upon TLR4. These cytokines and chemokines have known roles in antiviral immunity and highlight their roles in mediating survival in our model [52–59]. However, the impact of these individual cytokines/chemokines on the survival from SeV remains to be determined.

In summary, our study demonstrates that pretreatment with low dose LPS has the ability to prevent mortality from a severe respiratory viral infection. Further, this protection, which is mediated by TLR4-MyD88, appears to require macrophages but not neutrophils. This work demonstrates the potential for this LPS-TLR4-MyD88-macropahge axis as a potential therapeutic approach to limit the lethality of respiratory viral infections, expanding its role in immunopathology of disease. Our study is based on a mouse model, and future studies are needed to determine if similar mechanisms are operative in the human. Low doses of LPS (0.1 – 4 ng/kg) are used in several clinical trials to induce a transient inflammatory response [60] and test the efficacy of anti-inflammatory novel drugs during their early developmental stages in various diseases including atopy [61], showing potential human relevance for our study. The knowledge gained from this study may be applicable to other respiratory viral infections, particularly those from negative single stranded RNA viruses such as human parainfluenza viruses, HMPV, and RSV and could be leveraged in the future as a potential anti-viral therapeutic.

Supplementary Material

1

Key Points.

  1. Low dose LPS before but not after respiratory viral infection increases survival.

  2. LPS mediated survival depends upon macrophages but not neutrophils.

  3. LPS provides a survival advantage through type I IFN and TLR4-MyD88 signaling.

Acknowledgments

The authors acknowledge the critical help from the Flow Core at The Abigail Wexner Research Institute at Nationwide Children’s Hospital, Columbus OH (AWRINCH), and particularly Dave Dunaway for all the help troubleshooting flow cytometry runs and Bioplex assays, as well as Drs, Ross Maltz and Masako Shimamura at AWRINCH for the kind gift of Rag1−/− mice.

Funding sources:

This work was supported by the NIH R01 HL087778 (MHG), The Abigail Wexner Research Institute at Nationwide Children’s Hospital (MHG), the American Association of Immunologists Careers in Immunology Fellowship (MHG and JR), and the Alumni Grants for Graduate Research and Scholarship (AGGRS) at The Ohio State University (JR).

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