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. Author manuscript; available in PMC: 2017 Aug 1.
Published in final edited form as: Antiviral Res. 2016 Jun 14;132:131–140. doi: 10.1016/j.antiviral.2016.06.002

Signaling via pattern recognition receptors NOD2 and TLR2 contributes to immunomodulatory control of lethal pneumovirus infection

Tyler A Rice a,d,f, Todd A Brenner a,f, Caroline M Percopo a, Michelle Ma a, Jesse D Keicher b,e, Joseph B Domachowske c, Helene F Rosenberg a,g
PMCID: PMC4980231  NIHMSID: NIHMS797759  PMID: 27312104

Abstract

Pattern recognition receptors (PRRs) engage microbial components in the lung, although their role in providing primary host defense against respiratory virus infection is not fully understood. We have previously shown that Gram-positive Lactobacillus plantarum (Lp) administered to the respiratory tract promotes full and sustained protection in response to an otherwise lethal mouse pneumovirus (PVM) infection, a robust example of heterologous immunity. While Lp engages PRRs TLR2 and NOD2 in ex vivo signaling assays, we found that Lp-mediated protection was unimpaired in single gene-deleted TLR2−/− and NOD2−/− mice. Here we demonstrate substantial loss of Lp-mediated protection in a double gene-deleted NOD2−/−TLR2−/− strain. Furthermore, we demonstrate protection against PVM infection by administration of the bi-functional NOD2-TLR2 agonist, CL-429. The bi-functional NOD2-TLR2 ligand CL-429 not only suppresses virus-induced inflammation, it is significantly more effective at preventing lethal infection than equivalent amounts of mono-molecular TLR2 and NOD2 agonists. Interestingly, and in contrast to biochemical NOD2 and/or TLR2 agonists, Lp remained capable of eliciting primary proinflammatory responses from NOD2−/−TLR2−/− mice in vivo and from alveolar macrophages challenged ex vivo. Taken together, we conclude that coordinate engagement of NOD2 and TLR2 constitutes a key step in the genesis of Lp-mediated protection from a lethal respiratory virus infection, and represents a critical target for modulation of virus-induced inflammatory pathology.

Keywords: inflammation, cytokines, pattern recognition receptors, pneumovirus, probiotic

1. INTRODUCTION

Severe respiratory virus infections, including those caused by respiratory syncytial virus (RSV), are characterized by robust host inflammatory responses that, left unchecked, contribute significantly to illness severity (Mukherjee & Lukacs, 2013; Arruvito et al., 2015; Tabarani et al., 2013; Welliver, 2008). RSV, a virus of the family Paramyxoviridae, is an important pathogen that infects infants, children, and the elderly (Meissner, 2016; Walsh & Falsey, 2012); RSV-induced inflammation has also been linked to post-infection wheezing (Hall et al., 1984; Blanken et al., 2013). There is no vaccine against RSV, and monoclonal antibody prophylaxis is available only to a subset of infants considered to be at high risk for severe disease (Andabaka et al., 2013). Specific antivirals, including replication and fusion inhibitors, are currently in development (Gomez et al., 2014; De Clerq, 2015). Less attention has been directed towards therapeutic strategies designed to limit virus-induced inflammatory responses (Rosenberg & Domachowske, 2012).

We and others have explored inflammatory responses to and immunomodulatory control of respiratory virus infection using the pneumonia virus of mice (PVM) model of acute pneumovirus infection (Dyer et al., 2012; Bondue et al., 2011; Walsh et al., 2014; Scheer et al., 2014; Siegle et al., 2011). PVM is closely related to RSV; however, unlike RSV, PVM undergoes robust replication in vivo, most prominently in bronchial epithelial cells of inbred strains of mice. Acute PVM infection is accompanied by proinflammatory cytokine production and neutrophil recruitment to the airways (Bonville et al., 2006). By replicating the sequelae of a severe RSV infection, PVM provides a versatile and clinically relevant in vivo model for the evaluation of therapeutic strategies that promote immunomodulatory control of a detrimental host response (Rosenberg & Domachowske, 2012; Bem et al., 2011; Bonville et al., 2004).

Modulation of the host immune response by probiotic bacteria (e.g., Lactobacillus, Bifidobacteria) has been explored in depth, both in the clinical and translational literature, notably with respect to its role in the gastrointestinal tract (Oelschlaeger, 2010; Hevia et al., 2015). In mouse model studies, oral administration of probiotic bacteria results in the attenuation of inflammatory responses via strain-specific interactions with innate immune cells and with the gastrointestinal epithelium via engagement of Toll-like receptors (TLRs; Rakoff-Nahoum, et al., 2004; Koizumi et al., 2008; Liu et al., 2012) and nucleotide-binding oligomerization domain-containing protein (NOD)-like receptors (Wu et al., 2015; Macho Fernandez et al., 2011). However, at this time, the clinical efficacy of orally-administered probiotic bacteria with respect to respiratory virus infection and associated inflammation has not been clearly established (Hao et al., 2011; Esposito, et al., 2014; Ozen et al., 2015).

In an attempt to improve efficacy against respiratory virus infection, we and others have explored the outcome of Lactobacillus species administered locally, ie., directly to the respiratory tract (Gabryszewski et al., 2011; Izumo et al., 2010; Park et al., 2013; Tomasada et al., 2013). Our group has shown that L. plantarum (Lp), both live and heat-inactivated (in the latter form as a paraprobiotic (Taverniti & Guglielmetti, 2011)), when administered directly to the respiratory tract, results in robust and sustained protection against the lethal sequelae of PVM infection. We have shown that Lp is not primarily an antiviral agent (Gabryszewski et al., 2011); the beneficial impact of Lp-administration is primarily via suppression of local inflammation (Gabryszewski et al., 2011; Garcia-Crespo et al., 2013; Percopo et al., 2014; Percopo et al., 2015). Lactobacillus-mediated protection against respiratory virus infection is a profound example of heterologous immunity (also known in other contexts as trained immunity or innate memory (Levy & Netea, 2014; Netea et al., 2011; Wissinger et al., 2009; Locati et al., 2013; van der Meer et al., 2015) a state in which a host’s interaction with a primary infectious or inflammatory stimulus alters the immune response to a distinct, or unrelated agent.

In our previous work, we explored the receptor-mediated signaling processes elicited by Lp, and found that Lp specifically engages the pattern recognition receptors (PRRs) TLR2 and NOD2 in transfected HEK cells. However, and surprisingly, we found that Lp-mediated protection against lethal PVM infection was maintained in both TLR2−/− and NOD2−/− single gene-deleted mice (Percopo et al., 2015). Here, we utilize both NOD2−/−TLR2−/− double gene-deleted mice and a novel NOD2-TLR2 bifunctional ligand to demonstrate that the coordinate engagement of these PRRs contributes substantially to survival in response to an acute respiratory virus infection as well as to suppression of virus-induced inflammation.

2. MATERIALS AND METHODS

2.1 Mouse Strains

BALB/c mice were purchased from Charles River Laboratories (stock #555, Frederick, MD). C57BL/6, TLR2−/−, and NOD2−/− mice were purchased from The Jackson Laboratory; the gene-deleted strains were maintained as colonies in-house. All studies were performed in accordance with institutional guidelines provided by the NIAID Animal Use and Care Committee (ASP LAD8E). NOD2−/−TLR2−/− mice were generated by an initial parent cross (NOD2−/− x TLR2−/−) followed by cross-breeding of mixed heterozygote F1 generation to obtain full gene-deletion at both loci (Suppl. Fig. 1A). Genotyping strategies were as described for the parent strains by the Jackson Laboratory. Slightly fewer bone marrow cells per femur were recovered from adult NOD2−/−TLR2−/− mice (Suppl. Fig. 1B), although the lineage distributions were indistinguishable from the wild-type (data not shown). Likewise, although fewer splenocytes were isolated from NOD2−/−TLR2−/− mice (Suppl. Fig. 1C), the distribution of CD4+ T cells, CD8+ T cells, B cells, macrophages, DCs, neutrophils and eosinophils were indistinguishable from the wild-type (Suppl. Fig. 1D). NOD2−/−TLR2−/− mice breed normally and generate 5.2 ± 1.9 pups per litter.

2.2 Evaluation of bone marrow cells and splenocytes by flow cytometry

Bone marrow cells were collected from the femurs and tibiae of mice by flushing the opened bones with Iscove’s Modified Dulbecco’s medium (IMDM) as previously described (Dyer et al., 2008). Spleen cell suspensions in HBSS supplemented with 1% FBS and 10 mM HEPES were prepared as described (Dyer et al., 2011). Red blood cells were lysed with ACK lysing buffer (Lonza). Live/dead stain (Invitrogen) was added to the cells and non-specific antibody binding to Fc receptors was blocked with anti-mouse CD16/CD32 (BD Biosciences). For analysis of T and B cells, cell suspensions were incubated with anti-CD45-eF450 (eBioscience), anti-CD3-AF700 (eBioscience), anti-CD4-FITC (eBioscience), anti-CD8a-PE (BD), and anti-CD19-AF647 (BD) in phosphate-buffered saline with 0.1% bovine serum albumin (PBS/BSA) at 4°C for 30 min and washed with this buffer prior to analysis. For evaluation of myeloid cells, cell suspensions were incubated with anti-CD45-AF700 (BD), anti-CD11c-AF488 (BD), anti-SiglecF-PE (BD), anti-GR1-V450 (BD) and anti-MHCII-APC (eBioscience). At least 100,000 events were collected on an LSR II flow cytometer (BD Biosciences) and findings were analyzed in FlowJo 9.2.

2.3 Lactobacillus and biochemical pattern recognition receptor agonists

Lactobacillus plantarum (Lp; BAA-793) was grown overnight at 37°C in Mann-Rogosa-Sharpe (MRS) broth, heat-inactivated as previously described (Gabryszewski et al., 2011) and stored at −80°C at 1011 cells/mL. Heat-inactivated L. plantarum (Lp) was administered via intranasal inoculation to isoflurane-anaesthetized mice at 108 cells in 50 µL sterile PBS/BSA. Pattern recognition receptor agonists PAM3CSK4, muramyl-dipeptide (MDP) and the bifunctional NOD2-TLR2 agonist, CL-429 were purchased from Invivogen. For in vivo studies, CL-429 (stock solution 5 mg/mL in DMSO) was diluted in PBS/BSA and delivered to isoflurane-anesthetized mice as described together with the appropriate diluent controls (see Fig. 1A).

Figure 1. Lactobacillus plantarum (Lp)-mediated protection at the respiratory mucosa is impaired in NOD2−/−TLR2−/− mice.

Figure 1

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(A) Strategies used for administration of immunomodulatory agents to the respiratory tract prior to (on day −14 and day −7) or immediately after challenge (on day 1 and day 2) with a lethal inoculum of pneumonia virus of mice (PVM). These agents include heat-inactivated L. plantarum (Lp)) or the bifunctional NOD2-TLR2 agonist, CL-429. Survival of (B) wild-type (WT; C57BL/6) or (C) NOD2−/−TLR2−/− mice in response to Lp administered prior to PVM challenge as in (A); n = 7 – 14 mice per group, *p < 0.05, ***p < 0.001 vs. mice receiving no Lp (diluent control). (D) Percent and median survival of WT and NOD2−/−TLR2−/− treated with Lp or diluent control on day −14 and day −7 prior to PVM challenge on day 0; n = 7 – 14 mice per group; ***p < 0.001 between the two groups as shown.

2.4 Virus Infection

Titers of mouse-passaged stocks of pneumonia virus of mice (PVM) strain J3666 were determined by TCID50 assay (Percopo et al., 2011). Briefly, tumor fibroblast cells (ATCC CRL1542) were used as targets for serial ten-fold dilutions of mouse-passaged PVM stocks. Stocks were dialyzed (50 kDa molecular-weight cut-off) against tissue culture medium to remove cell-activating and cytotoxic mediators prior to evaluation. Cytopathic effect (CPE), which includes rounding up and piling of the target fibroblasts, was evaluated at day 3 after exposure to virus, and the TCID50 value was calculated by standard Reed-Muench methodology. Infections were established in isoflurane-anaesthetized mice via intranasal inoculation with 0.2 TCID50 units (to BALB/c mice) or 2 TCID50 units (to C57BL/6 mice and the gene-deleted mice, which are on the C57BL/6 background) in 50 µL Dulbecco’s Modified Eagle’s medium diluent via strategies as shown in Fig. 1A.

2.5 Airway leukocytes, virus titer, proinflammatory cytokines and lung tissue histology

Mice were sacrificed at the time points indicated after inoculation with Lp alone, or Lp followed by PVM infection (see Fig. 1A). Bronchoalveolar lavage (BAL) was performed (0.8 mL x 2) with PBS/BSA. Cytospin slides were prepared and stained (DiffQuik) for total and differential leukocyte counts and a minimum 200 cells per mouse were scored. Virus titers in lung tissue of PVM-infected mice was measured by dual-standard curve quantitative RT-PCR with primers, probes and standard curves as previously described in detail (Percopo et al., 2014b; Gabryszewski et al., 2011). Briefly, cDNAs are prepared from total lung RNA from mice infected with PVM. Both virus and cellular RNAs are transcribed into cDNAs, and quantitated with specific primer-probes. For PVM, virus genomic RNA is measured by targeting the PVM-specific small hydrophobic (SH) gene, and the values are reported as copy number, a value determined by interpolating to a standard curve. These values are normalized to copy number of the cellular housekeeping gene, GAPDH, which is also determined by interpolating to a standard curve. Virus titer is reported as copy number of the amplified virus gene (PVMSH) divided by the copy number of GAPDH. Cytokine ELISAs (DuoSets, R&D systems) were performed on clarified lung tissue homogenates and corrected for total protein by BCA assay (Pierce). Tissue sections prepared from 10% phosphate-buffered formalin-fixed lung tissue were stained with hematoxylin and eosin (H&E; Histoserv, Inc., Germantown, MD).

2.6 Alveolar macrophage isolation and ex vivo stimulation

Alveolar macrophages (AMs) were isolated from naive mice (WT and NOD2−/−TLR2−/−) by BAL with 4 mL sterile PBS/BSA. In experiments with Lp, cells from BAL were washed 2 times with sterile PBS/BSA and were plated (3 × 105 cells / mL) in 1 mL complete media (RPMI 1640 with 10% fetal bovine serum, 2 mM L-glutamine and penicillin/streptomycin); Lp was added to a final concentration of 108 cells /mL. After 4 hrs, the adherent monolayer was washed twice with sterile PBS/BSA, then cultured overnight in 1 mL complete medium at 37°C with 5% CO2. In experiments with PRR agonists, cells from BAL were washed as above and 3 × 105 cells per well were plated in 1 mL complete medium. AMs were allowed to adhere for 4 hrs then washed with PBS/BSA as above. Agonists Pam3CSK4, MDP, or CL-429 were added to final concentrations of 100 ng/mL, 10 µg/mL, or 10 µg/mL, respectively, and cultures were incubated overnight as above. Cytokine ELISAs (DuoSet, R&D Systems) were performed on primary AM culture supernatants as per manufacturer’s instructions.

2.7 qPCR analysis of TLR2 and NOD2 expression in AMs

AMs were isolated from naive and Lp-primed WT C57BL/6 and NOD2−/−TLR2−/− mice as described above; RNA was isolated using the RNeasy mini kit (Qiagen). Absolute copy numbers of transcripts encoding TLR2 and NOD2 were determined with transcript-specific primer/probes (Mm00442346_m1 for TLR2; and Mm00467543_m1 for NOD2; Advanced Biotechnologies, Inc.). The standard curves included a 389 bp insert spanning exon 6 of the transcript encoding mouse Nod2, cloned into plasmid PCR 2.1. The plasmid including the gene encoding mouse TLR2 was obtained from AddGene (cat #13083). The primer/probes and standard curve for GAPDH were as described previously (Percopo et al., 2014b).

2.8 Statistical Analysis

Data were analyzed using Mann-Whitney u-test, Log-Rank test, and 1-way Analysis of Variance (ANOVA) as appropriate via algorithms within GraphPad 6.0. Error bars represent standard deviation unless otherwise indicated.

3. RESULTS

3.1. Signaling via NOD2 and TLR2 contributes to Lp-mediated survival

Our previous work revealed that administration of L. plantarum (Lp) to the respiratory tract either several weeks prior to or immediately after (within 36 hours of) virus challenge protected mice against the lethal sequelae of acute PVM infection (strategies diagrammed in Fig. 1A (Gabryszewski et al., 2011; Percopo et al., 2015)). Although Lp signals through NOD2 and TLR2 when each is expressed individually in transfected HEK cell lines (Percopo et al., 2015), we found that PVM-infected single gene-deleted NOD2−/− or TLR2−/− mice remained protected by administration of Lp to the respiratory tract (Percopo et al., 2015).

As shown in Fig. 1B, administration of Lp to the respiratory tract (“priming”) of wild-type mice on day −14 and day −7 prior to virus challenge on day 0 resulted in full protection against the lethal sequelae of PVM infection. The critical roles of TLR2 and NOD2 were revealed in response to priming of mice devoid of both pattern recognition receptors (NOD2−/−TLR2−/−), in which substantial protection against subsequent virus infection was lost (100%, vs. 20% survival, ***p < 0.001; Fig. 1C and 1D]. Of note, Lp-primed NOD2−/−TLR2−/− mice still exhibit prolonged survival when compared to their control (no Lp)-primed gene-deleted counterparts (median survival 11 vs. 8 days, *p < 0.04; Fig. 1C). While these findings suggest the existence of additional Lp-mediated protective mechanisms (see Discussion), our results indicate that signaling via NOD2 and TLR2 is clearly critical to elicit full protection.

3.2. The bifunctional NOD2-TLR2 ligand CL-429 promotes survival via immuno-modulatory control of lethal pneumovirus infection

The bi-functional ligand, CL-429, includes covalently-linked murabutide and bis-palmitoyl lipid moieties that activate both NOD2 and TLR2 receptors, respectively (Pavot et al., 2014). As shown in Fig.2A, CL-429 promotes survival when administered post-virus challenge (see Fig. 1A); fifty percent (50%) of the wild-type mice inoculated with PVM on day 0 and with CL-429 (12.5 µg/dose) on days 1 and 2 survive, compared to mice receiving diluent alone (***p < 0.001). As anticipated, no responses to CL-429 were detected in NOD2−/−TLR2−/− mice.

Figure 2. Bifunctional TLR2-NOD2 agonist CL-429 administered to the respiratory mucosa also elicits protection against acute respiratory virus infection.

Figure 2

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(A) Survival of wild-type and NOD2−/−TLR2−/− mice inoculated with PVM on day 0 followed by CL-429 (12.5 µg per dose) or diluent on days 1 and 2 (post-exposure; see Fig. 1A); n = 10 – 11 mice per group, ***p < 0.001 vs. all other survival curves shown. (B) Survival of wild-type (BALB/c) mice in response to priming with CL-429 (2.5 µg per dose) prior to challenge with PVM on day 0; n = 10 – 20 mice per group, **p < 0.01 vs. diluent or MDP alone (2.5 µg per dose), ***p < 0.001 vs. Pam3CSK4 alone (2.5 µg / dose) or Pam3CSK4 + MDP (2.5 µg each per dose). (C) Survival of wild-type (BALB/c) mice in response to priming with CL-429 (2.5 µg per dose) administered on day −14 and day −7 prior to challenge with PVM on day 19 (see Fig. 1A); n = 5 mice per group, **p < 0.01 vs. diluent alone.

Priming of the respiratory mucosa of with CL-429 (2.5 µg/dose on days −14 and day −7, see Fig. 1A) prior to PVM challenge also resulted in substantial survival (90%) of wild-type BALB/c mice (***p < 0.001 vs. diluent; Fig. 2B). While priming with the NOD2 agonist, MDP alone had no impact on survival at this dose, priming with the TLR2 agonist, Pam3CSK4 or equivalent amounts of the two ligands together resulted in 35% and 40% survival respectively (**p < 0.01 vs. diluent or MDP alone). Interestingly, survival in response to the bifunctional TLR2-NOD2 agonist CL-429 was significantly more robust than the response to equivalent amounts of individual or combined TLR2 and NOD2 agonists (*p < 0.001; see Discussion).

Similar to the sustained survival observed in response to priming with Lp [31, 36], priming with CL-429 also results in sustained protection against acute respiratory virus infection. As shown in Fig. 2C, we observe 80% survival in CL-429-primed mice when PVM infection is delayed from day 0 until day 19, nearly 4 weeks after the final inoculation with the NOD2-TLR2 ligand [Fig. 2C].

3.3. Virus titers, proinflammatory cytokine production and neutrophil recruitment in Lp-primed PVM-infected wild-type and NOD2−/−TLR2−/− mice

Our earlier studies documented the impact of Lp-priming on survival, virus replication and virus-induced proinflammatory responses (Gabryszewski et al., 2011; Garcia-Crespo et al., 2013; Percopo et al., 2014a; Percopo et al., 2015). Here, we compare the responses wild-type of NOD2−/−TLR2−/− mice to Lp-priming followed by PVM infection. As shown in Fig. 3A, Lp-priming of wild-type mice results in diminished virus recovery, a response that is not observed in Lp-primed NOD2−/−TLR2−/− mice (**p < 0.01 vs. WT). Similarly, Lp-priming results in suppression of neutrophil recruitment in PVM-infected wild-type mice, but not PVM-infected NOD2−/−TLR2−/− mice (Fig. 3B). Lung tissues from Lp-primed, PVM-infected wild-type and NOD2−/−TLR2−/− mice are shown in Fig. 3C and Fig. 3D, respectively. Consistent with the results shown in Fig. 3B, the lungs of Lp-primed, PVM-infected NOD2−/−TLR2−/− mice display significant alveolitis, with neutrophil recruitment and accumulation in the large and small airways. In contrast, the lungs of Lp-primed PVM-infected wild-type mice remain comparatively clear of neutrophilic inflammation. Likewise, Lp-primed, PVM-infected NOD2−/−TLR2−/− mice exhibit limited suppression of virus-induced IL-6 (Fig. 3E) and no significant suppression of CCL2 (Fig. 3F) or CXCL10 (Fig. 3G).

Figure 3. Lp does not suppress virus-induced inflammatory responses in NOD2−/−TLR2−/− mice.

Figure 3

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Mice were Lp-primed at day −14 and day −7 and were inoculated with PVM at day 0 (see Fig. 1). (A) Virus titer in lung tissue at day 7; **p < 0.01; ***p < 0.001; ns, not significant (B) Percent neutrophils in bronchoalveolar lavage (BAL) fluid on day 7 as described in (A); n = 5 – 9 mice per group; *p < 0.05, **p < 0.01. Lung tissue from (C) WT and (D) NOD2−/−TLR2−/− mice primed with Lp prior to PVM infection, and evaluated on day 7, with filled arrow denoting neutrophils in the airways; original magnification 20X. Proinflammatory cytokines in lung tissue of mice on day 7 as described in (A), including (E) IL-6, (F) CCL2 and (G) CXCL10; n = 5 – 7 mice per group, *p < 0.05, **p < 0.01 vs. +Lp; ǂp < 0.05 vs. WT; ǂǂ p < 0.01 vs. WT. All values shown are ± standard deviation.

3.4 Suppression of proinflammatory cytokine production in response to CL-429

As we have observed in response to Lp using this inoculation strategy (Percopo et al., 2015), survival in response to CL-429 [Fig. 4A] is also associated with diminished virus recovery [Fig. 4B], and significant suppression of virus-induced proinflammatory cytokines in lung tissue, including IL-6 [Fig. 4C], CCL2 [Fig. 4D], CXCL10 [Fig. 4E] and CXCL1 [Fig. 4F].

Figure 4. CL-429 administered to the respiratory mucosa suppresses virus-induced proinflammatory cytokine responses.

Figure 4

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Wild-type (BALB/c) mice inoculated with PVM on day 0 (black arrow) followed by CL-429 or diluent on days 1 and 2 (A) Survival (2.5 or 25 µg per dose) vs. diluent, n = 5 – 10 mice per group, ***p < 0.001 (B) Virus titers in lung tissue on day 5, CL-429 (2.5 µg/dose) vs. diluent; n = 11 – 12 mice per group; **p < 0.01. Proinflammatory cytokines (C) IL-6 (D) CCL2 (E) CXCL10 and (F) CXCL1 in lung tissue on day 5, (2.5 µg per dose) vs. diluent; n = 5 – 7 mice per group, *p < 0.05, **p < 0.01. All values shown are ± standard deviation.

3.5 Lp, but not CL-429, can elicit primary proinflammatory responses in NOD2−/−TLR2−/− mice

One of the primary principles of heterologous immunity (also known as innate memory or trained memory; see Discussion) is that the innate immune system alters its responses after recovering from a primary infectious or inflammatory stimulus (Levy & Netea, 2014; Netea et al., 2011; Wissinger et al., 2009; Locati et al., 2013; van der Meer et al., 2015). We have shown previously that Lp-priming alone results in local expression of numerous proinflammatory cytokines and recruitment of neutrophils to the lung tissue within hours of the primary stimulus; this response is transient and neutrophils and cytokine levels return to baseline within 24 hrs [36]. As shown in Fig. 5, despite gene-deletion of NOD2 and TLR2, Lp elicits a significant primary inflammatory response in the lungs of the double gene-deleted NOD2−/−TLR2−/− mice. At four hrs after instillation, elevated levels of IL-6 [Fig. 5A], CCL2 and CXCL1 (data not shown) and recruited neutrophils [Fig. 5B] were detected in lung homogenates and BAL fluid, respectively, of NOD2−/−TLR2−/− mice, to an extent indistinguishable from the wild-type. We examined this issue further by evaluating the responses of airway macrophages (AMs) isolated from BAL from wild-type and NOD2−/−TLR2−/− mice. Ninety percent (90%) of these cells were CD45+Siglec F+CD11c+, the characteristic immuno-phenotype of resident alveolar macrophages (Hussell & Bell, 2014). AMs from wild-type mice express both TLR2 and NOD2; expression of NOD2 increases in response to priming with Lp [Suppl. Fig. 2]. As shown in Fig. 5C, isolated AMs from wild-type mice generate IL-6 in response to challenge with the TLR2 ligand, Pam3CSK4, the NOD2 ligand, MDP, and the bifunctional TLR2-NOD2 ligand, CL-429; AMs from NOD2−/−TLR2−/− mice do not respond to any of these agonists. In contrast, AMs from NOD2−/−TLR2−/− mice produce IL-6 in response to challenge with Lp as do AMs from wild-type mice [Fig. 5D]. As noted earlier, while our results indicate that signaling via NOD2 and TLR2 is a critical element underlying Lp-mediated protection, Lp remains capable of engaging other receptors and additional signaling mechanisms in vivo (considered further in the Discussion).

Figure 5. Lp but not CL-429, elicits primary inflammatory responses from NOD2−/−TLR2−/− mice.

Figure 5

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(A) IL-6 detected in whole lung tissue homogenates from WT and NOD2−/−TLR2−/− mice at t = 4 hrs after Lp-challenge of the respiratory tract; n = 4 mice per group, **p < 0.01 vs. no Lp diluent control for each strain. (B) Percent neutrophils in BAL fluid of mice challenged with Lp as in (A); (C) IL-6 in supernatants of airway macrophage cultures from WT and NOD2−/−TLR2−/− mice challenged ex vivo with Pam3CSK4 (100 ng/mL), MDP (10 µg/mL) or CL-429 (10 µg/mL); **p < 0.01 vs. responses of airway macrophages from NOD2−/−TLR2−/− mice. (D) Same cultures as in (C) challenged ex vivo with Lp (108 cells/mL); for (C) and (D), n = 5, ***p < 0.001 vs. the responses of untreated airway macrophages. All values shown are ± standard deviation.

4. DISCUSSION

In this manuscript, we continue our exploration of the clinically benign Gram-positive microorganism, Lactobacillus plantarum (Lp) and its role in preventing the detrimental sequelae of respiratory virus infection. Here, we define a receptor-dependent mechanism via which Lp administered to the respiratory mucosa elicits heterologous immunity against lethal PVM infection.

Heterologous immunity, also known as trained immunity or innate memory, is a concept that focuses on the way(s) in which the immune system responds differently to a distinct (or heterologous) organism after a primary infectious or inflammatory stimulus. While there are numerous examples of this principle described in the literature, the trained immune responses elicited systemically in response to mycobacteria are among the best characterized. Several groups have noted that mice vaccinated with Bacille Calmette Guérin (BCG) developed resistance to antigenically-unrelated bacterial, fungal and parasitic infections (reviewed in van der Meer, et al. 2015); similar observations were made among BCG-vaccinated children (Garly et al., 2003). Netea and colleagues (van der Meer et al., 2015; Ifrim et al., 2014) have identified NOD2 as the critical receptor underlying systemic responses to BCG; similar dectin-1-dependent mechanisms promote trained immunity secondary to systemic exposure to fungal antigens.

By contrast, receptor-mediated mechanisms underlying heterologous immunity in the lung remain to be clearly elucidated. We showed earlier that Lp-mediated protection against PVM infection remained unimpaired in single gene-deleted NOD2−/− and TLR2−/− mice. Generation of double gene-deleted NOD2−/−TLR2−/− mice has permitted us to identify the contributions of these PRRs to Lp-mediated responses. The roles of TLR2 and NOD2, their interactions, cross-regulation, and involvement with inflammatory disease have been characterized most extensively in the gut micro-environment where they serve to regulate the development of antigen-dependent colitis (Watanabe et al., 2006; Yang et al., 2007). In the lung, independent responses of TLR2 and NOD2, and their interactions with bacterial respiratory pathogens have been characterized (Yu et al., 2014; Leissinger et al., 2014). Of particular note, Davis and colleagues (2011) found that clearance of the Streptococcus pneumoniae pathogen from the upper respiratory tract was impaired only upon ablation of both TLR2 and NOD2. Interestingly, the effective response to S. pneumoniae was dependent on CCL2/CCR2-dependent recruitment of proinflammatory monocyte / macrophages. In contrast, we have found that Lp not only suppresses CCL2, protection elicited by Lp is fully effective in CCR2−/− mice and may be mediated by a subset of monocytes that are devoid of this receptor (Brenner & Rosenberg, ms. in preparation). More recently, Dorrington and colleagues (2013) found that the class A scavenger receptor MARCO is required for TLR2 and NOD2-mediated clearance of S. pneumoniae. Interestingly, MARCO is one of the several receptors implicated in macrophage-mediated “adaptive” or heterologous responses (Bowdish et al., 2007).

Equally intriguing, we find that Lp remains capable of eliciting primary inflammatory responses in vivo in NOD2−/−TLR2−/− gene-deleted mice. While assays performed in vitro with HEK transfectants suggested that NOD2 and TLR2 alone were capable of interacting with Lp (Percopo et al., 2015), these assays by nature evaluate responses of single PRRs in isolation. In vivo, mice may compensate for the absence of both NOD2 and TLR2 by developing alternative signaling mechanisms to detect Gram-positive bacteria and/or may rely more heavily on contributions from other binding proteins, such as peptidoglycan recognition proteins (PGLYRPs; Royet et al. 2011). The observation that Lp remains capable of generating a primary inflammatory response in NOD2−/−TLR2−/− mice may explain the relatively small but still statistically significant protection elicited by Lp in this mouse strain.

Complementing our studies with gene-deleted mice, we found that the bifunctional NOD2-TLR2 ligand CL-429, also elicits protection against lethal respiratory virus infection when administered to the respiratory tract. Specifically, we show for the first time that the bi-functional NOD-TLR2 ligand CL-429 suppresses virus-induced inflammation in vivo and prevents the lethal sequelae of acute respiratory virus infection. This bifunctional agonist, generated by Pavot and colleagues (2014), engages both TLR2 and NOD2 receptors, and was shown to promote augmented production of antigen-specific Igs in vivo over and above the responses to monomolecular NOD2 and TLR2 ligands administered together. The complementary features linking the adjuvant activity reported by Pavot and colleagues (2014), and our observations, which relate to the acute and prolonged suppression of detrimental virus-induced inflammation are intriguing to consider. Of note, we have shown clearly that engagement of NOD2/TLR2 receptors by Lp suppresses virus-induced inflammation and provides long-term protection against the lethal sequelae of acute respiratory infection in mice that are devoid of B cells and immunoglobulins (Percopo et al., 2014a). However, we have also found that CL-429 and Lp provide only short-term protection in mice that are unable to clear virus (Rosenberg, Percopo and Ma, manuscript in preparation). Overall, the possibility that CL-429 can reduce virus-induced inflammation while at the same time augmenting adaptive immune responses that facilitate virus clearance suggests significant multi-tiered clinical utility worthy of further development.

Also analogous to the findings by Pavot and colleagues (2014), we found that bifunctional CL-429 is more effective than equivalent concentrations of independent, single NOD2 and TLR2 agonists, administered alone or together. The specific details underlying this improved functionality also await future study. Among the possibilities, covalent linkage between MDP and the TLR2 agonist may provide a more favorable conformation the murabutide, such as one that coordinates a more favorable receptor co-localization (see, for example, Müller-Anstett et al., 2010). Another possibility, the covalent linkage to MDP may improve the conformation and the receptor-binding efficacy of the TLR2 agonist.

Several groups have explored the role of PRR ligands in promoting immunity to respiratory virus infection. Among them, Wong and colleagues (2005) explored the impact of liposomal delivery of nucleotide agonists (poly I:C and CpG-oligonucleotides, or ODNs, which are TLR3 and TLR9 agonists, respectively), which provided significant protection against influenza infection. Tuvim and colleagues (2012) examined responses to Pam2CSK4 together with ODNs and observed synergy between these agonists against lethal influenza infection. A more recent study by Drake and colleagues (2013) extended the antiviral impact of TLR2/6/9 agonists to include parainfluenza virus. Furthermore, Shafique and colleagues (2013) have incorporated Pam3CSK4 and L18-MDP into virosomes prepared from the human RSV pathogen, and reported increased systemic and mucosal antibody responses to live virus challenge.

Our results, taken together, demonstrate for the first time that coordinate TLR2-NOD2 engagement is crucial in order to elicit the full anti-inflammatory, protective phenotype associated with Lp treatment.

5. CONCLUSIONS

In summary, the work presented here reveals critical receptor-dependent mechanisms by which Gram-positive Lactobacillus plantarum engages host cells at the respiratory mucosa in order to elicit heterologous immunity against lethal pneumovirus infection. We have shown previously that administration of Lp to the respiratory tract resulted in both immediate and sustained protection against the lethal sequelae of acute PVM infection. We find here that Lp-mediated protection against lethal virus infection is lost specifically in NOD2−/−TLR2−/− mice, resulting in augmented virus recovery and local inflammatory responses relative to Lp-treated wild-type mice. Furthermore, administration of a specific NOD2-TLR2 bi-functional ligand also results in protection against lethal respiratory virus infection. Taken together, we have identified PRRs NOD2 and TLR2 as contributing the Lp-mediated heterologous immunity and as critical targets for modulation of virus-induced immunopathology.

Supplementary Material

3. Suppl. Fig. 1. Characterization of NOD2−/−TLR2−/− mice.

A. PCR-genotyping of wild-type (+/+), heterozygous TLR2+/−/NOD2+/− F1 mice, and two examples (#6152 and #6200) of mice homozygous for the gene-deletion at both loci (NOD2−/−TLR2−/−). B. Total bone marrow cells from femurs of WT and NOD2−/−TLR2−/− mice. C. Total splenocytes isolated from WT and NOD2−/−TLR2−/− mice; D. Percent of total splenocytes from WT and NOD2−/−TLR2−/− mice represented by CD4+ T cells (CD3+CD4+) CD8+ T cells (CD3+CD8+) B cells (CD19+) monocyte-macrophages (MΦ, CD11c+Gr1), dendritic cells (DC, CD11c+MHCII+Gr1), polymorphonuclear leukocytes (pmn, neutrophils, CD11cGr1+) and eosinophils (eos, CD11cGr1SiglecF+) as a percent of total splenocytes.

4. Suppl. Fig. 2. Expression of pattern recognition receptors in alveolar macrophages from naïve wild-type C57BL/6 mice.

A. NOD2; B. TLR2, as determined by dual standard curve quantitative RT-PCR as described in Methods.

Highlights.

  • Lp-mediated protection against lethal PVM infection is impaired in NOD2−/−TLR2−/− mice.

  • Protection against PVM infection can be elicited by administration of the bifunctional NOD2-TLR2 agonist CL-429.

  • Both Lp and CL-429 suppress virus-induced inflammation at the respiratory mucosa.

  • Lp, but not CL-429, can elicit primary inflammatory responses from NOD2−/−TLR2−/− mice.

Acknowledgments

This manuscript is dedicated to Dr. Kimberly D. Dyer and include our wishes for her speedy recovery. The work herein was supported by funds from NIAID Division of Intramural Research (AI000943) to HFR.

Abbreviations

PVM

pneumonia virus of mice

Lp

Lactobacillus plantarum

RSV

respiratory syncytial virus

PRR

pattern recognition receptor

Footnotes

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Supplementary Materials

3. Suppl. Fig. 1. Characterization of NOD2−/−TLR2−/− mice.

A. PCR-genotyping of wild-type (+/+), heterozygous TLR2+/−/NOD2+/− F1 mice, and two examples (#6152 and #6200) of mice homozygous for the gene-deletion at both loci (NOD2−/−TLR2−/−). B. Total bone marrow cells from femurs of WT and NOD2−/−TLR2−/− mice. C. Total splenocytes isolated from WT and NOD2−/−TLR2−/− mice; D. Percent of total splenocytes from WT and NOD2−/−TLR2−/− mice represented by CD4+ T cells (CD3+CD4+) CD8+ T cells (CD3+CD8+) B cells (CD19+) monocyte-macrophages (MΦ, CD11c+Gr1), dendritic cells (DC, CD11c+MHCII+Gr1), polymorphonuclear leukocytes (pmn, neutrophils, CD11cGr1+) and eosinophils (eos, CD11cGr1SiglecF+) as a percent of total splenocytes.

NIHMS797759-supplement-3.tif (516.4KB, tif)
4. Suppl. Fig. 2. Expression of pattern recognition receptors in alveolar macrophages from naïve wild-type C57BL/6 mice.

A. NOD2; B. TLR2, as determined by dual standard curve quantitative RT-PCR as described in Methods.

NIHMS797759-supplement-4.tif (196.2KB, tif)

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