OM-85 is an immunomodulator of interferon-β production and inflammasome activity

The inflammasome–IL-1 axis and type I interferons (IFNs) have been shown to exert protective effects upon respiratory tract infections. Conversely, IL-1 has also been implicated in inflammatory airway pathologies such as asthma and chronic obstructive pulmonary disease (COPD). OM-85 is a bacterial extract with proved efficacy against COPD and recurrent respiratory tract infections, a cause of co-morbidity in asthmatic patients. We therefore asked whether OM-85 affects the above-mentioned innate immune pathways. Here we show that OM-85 induced interferon-β through the Toll-like receptor adaptors Trif and MyD88 in bone marrow-derived dendritic cells. Moreover, it exerted a dual role on IL-1 production; on the one hand, it upregulated proIL-1β and proIL-1α levels in a MyD88-dependent manner without activating the inflammasome. On the other hand, it repressed IL-1β secretion induced by alum, a well-known NLRP3 activator. In vivo, OM-85 diminished the recruitment of inflammatory cells in response to peritoneal alum challenge. Our findings therefore suggest that OM-85 favors a protective primed state, while dampening inflammasome activation in specific conditions. Taken together, these data bring new insights into the mechanisms of OM-85 action on innate immune pathways and suggest potential explanations for its efficacy in the treatment of virus-induced airway diseases.

From the medical standpoint, recurrent respiratory tract infections (RTIs), COPD, as well as wheezing and asthma are major public health problems. Concomitantly, prevalence of antibiotic resistance among common respiratory pathogens has recently risen, highlighting the need to develop strategies of immunization against respiratory bacteria and viruses [40][41][42] . Launched more than 30 years ago, OM-85 (Broncho-Vaxom ® , Broncho-Munal ® , Ommunal ® , Paxoral ® , Vaxoral ® ), an oral medicine of biological origin used for the prevention of recurrent RTIs and/or exacerbations in at-risk populations, was shown to be effective and safe in both children and adults [43][44][45][46][47] . Its active principle, an extract of bacterial lysates isolated from 21 known respiratory pathogenic strains, showed protection against airway infections of bacterial and viral origin. In particular, several studies highlighted the capacity of OM-85 to trigger immunomodulatory and protective immune responses against diverse pathogens in vivo [48][49][50][51][52][53][54] , including influenza and respiratory syncytial virus as well bacterial superinfection following influenza 55,56 .
Despite these findings, the effects of OM-85 on the production of type I interferons or inflammasome-mediated IL-1 production have not been assessed. We thus dissected the role of OM-85 in regulating these pathways in vitro. We found that OM-85 induced IFN-β production and acted as an effective priming agent inducing substantial levels of proIL-1α and β . Surprisingly, OM-85 also interfered with inflammasome activity specifically in response to alum. These results suggest that OM-85 dampens selected inflammatory reactions, while potentiating innate immune defenses, two effects that might contribute to reducing infections and alleviating COPD and asthmatic conditions in the context of the respiratory tract.

Results
OM-85 induces interferon-β production. Given the potential relevance of type I IFN in asthmatic patients, we investigated the ability of OM-85 to induce this cytokine. We focused on DCs, which are proficient in producing type I IFNs and in activating the inflammasome pathway 21,25,26 . We therefore treated bone marrow-derived DCs (BMDCs) for 16 hours and assessed IFN-β release in the supernatant. IFN-β production was detected and followed a bell-shaped curve as a function of OM-85 concentration (Fig. 1a). To gain insights into the OM-85-dependent mechanisms inducing IFN-β , we took advantage of Trif-and MyD88-deficient BMDCs. Interestingly, the absence of either Trif or MyD88 drastically affected OM-85-driven IFN-β production (Fig. 1b). Taken together, we show here that OM-85 has the ability to induce IFN-β production, an important cytokine playing immunomodulatory and antiviral effects, in a MyD88-and Trif-dependent manner.
OM-85 does per se not lead to IL-1 release. We next tested the effects of OM-85 on the inflammasome/IL-1 axis. Given the complex nature of OM-85, we first asked whether this extract exhibited the capacity to induce IL-1β and α production. This encompasses the steps of inducing their precursor forms, referred to as "priming", and of activating the inflammasome for their cleavage and/or secretion. We therefore treated BMDCs with increasing doses of OM-85 for either 6 or 16 hours and measured the release of IL-1β and IL-1α by ELISA. As shown in Fig. 2a and b, no substantial production of these cytokines was observed as compared to the unstimulated condition, whereas they were nicely released upon standard two-step stimulation performed by LPS-mediated priming and the NLRP3 inflammasome activator aluminium (alum). This indicates that OM-85 has not per se the capacity to induce the inflammasome/IL-1 cascade.
OM-85 acts as a priming signal mainly through MyD88. Despite the inability to induce IL-1 secretion, the bacterial origin of OM-85 suggested it to be able to act as a priming signal. We therefore tested its ability to prime BMDCs, which were then treated with alum. As shown in Fig. 3a and b, exposure to OM-85 effectively induced the production of IL-1β and IL-1α by alum-triggered BMDCs. We thus tested the induction of proIL-1β . IFN-β secretion was measured by ELISA. Results represent mean ± SD (n = 3 technical replicates) and are representative of at least four (a) and two (b) independent experiments. Statistical differences were calculated between the condition without OM-85 (0) and each of the following doses using Student's t-test adjusted by Bonferroni correction over 5 (a) and between the wild type condition and the mutant genotypes using Student's t-test adjusted by Bonferroni correction over 2 (b). ns, non-significant; ***p ≤ 0.001.
SciEntific RepoRts | 7:43844 | DOI: 10.1038/srep43844 and proIL-1α in BMDCs upon a four hours treatment with OM-85. As shown in Fig. 3c, the bacterial extract increased the amount of proIL-1α and proIL-1β in a dose-dependent manner.
Next, we used Trif-and MyD88-deficient BMDCs to understand whether these adaptor proteins mediated OM-85-induced proIL-1β and proIL-1α . Whereas the absence of Trif only slightly affected the levels of proIL-1α , MyD88 deficiency abolished OM-85-induced priming with regard to both proIL-1β and proIL-1α (Fig. 3c). These data indicate that OM-85 did not deploy toxic effects even at high concentrations and was active, modulating pro-IL-1β and α in a MyD88-dependent manner.
OM-85 acts as a priming signal for the NLRP3 and the AIM2 inflammasomes. We next asked whether OM-85 was an effective priming agent for multiple activators of the NLRP3 as well as for the AIM2 inflammasome, as both these platforms are important for detection of and protection from viral infections. To address these questions, BMDCs pre-treated for four hours with different doses of OM-85 were exposed to the particulate NLRP3 activators alum and monosodium urate (MSU) crystals as well as to the soluble activators ATP and nigericin. As illustrated in Fig. 4a and b, OM-85 was an efficient priming signal for all of these stimuli. While the effects by OM-85 treatment were consistent in all experiments, it is important to mention that the activity of OM-85 on BMDC was subject to some variation, as illustrated here between panels 3a/b and 4a/b at the lower doses applied (e.g. 10 ug/ml).
In addition, we observed that OM-85 acted in the same way for the AIM2 inflammasome ( Fig. 4c and d). Intriguingly however, BMDCs pre-treated with the highest tested dose of OM-85 showed reduced production of IL-1α and IL-1β upon polydA:dT-mediated activation.

OM-85 pre-treatment interferes with LPS and alum-induced inflammasome activation.
The observation that high-dose OM-85 induced a decrease of AIM2-mediated IL-1α and IL-1β release prompted us to further explore potential dampening effects of OM-85 on inflammasome activity. We therefore performed a modified version of the inflammasome activation assay in which we pre-treated BMDCs overnight with OM-85 and added LPS for four hours, followed by inflammasome activators. Whereas we did not observe significant inhibitory effects on IL-1α and IL-1β secretion upon inflammasome triggering by ATP, we found that IL-1β secretion induced by alum and -to a smaller extent by MSU -was downmodulated by OM-85 pre-treatment ( Fig. 5a and Supplementary Figure 1a). However, secreted IL-1α was not substantially decreased, as shown in Fig. 5b. In order to explain the reduced release of IL-1β , we checked the levels of cleaved caspase-1. Interestingly, caspase-1 activation was decreased by overnight OM-85 pre-treatment ( Fig. 5c and Supplementary Figure 1b). Taken together, these data suggests that OM-85 pre-treatment dampens IL-1β production, at least partly through reducing inflammasome activity in response to alum.
Alum-driven peritonitis is reduced by OM-85. Based on the previous finding, we then tested whether the immunomodulatory effects of OM-85 on the inflammasome were recapitulated in an in vivo model. We focused on the well-established alum-dependent peritonitis model, which is mediated by IL-1α and IL-1β and is commonly used to assess inflammasome activity in vivo 19 . Wild type mice were pre-treated intravenously with OM-85, or PBS as control and, five hours later, injected intraperitoneally (i.p.) with alum. Twelve hours post alum-injection, mice were sacrificed and peritoneal exudate cells analyzed by flow cytometry. As depicted in Fig. 6a, a remarkable decrease in the numbers of recruited cells was observed. In addition, when specifically assessing the abundance of individual inflammatory subsets, a significant decrease in the numbers of neutrophils, eosinophils, as well as macrophages was observed (Fig. 6b-d). These data indicate that OM-85 has the ability of interfering also in vivo with alum-induced inflammatory reactions.  (a and b) Dose-response and time course (6 and 16 h) for the release of IL-1β (a) and IL-1α (b) by BMDCs in response to the indicated concentrations of OM-85 as measured by ELISA. As control, stimulation was performed with LPS (100 ng/ml) for 6 and 16 h or LPS (100 ng/ml) followed by addition of alum (300 μ g/ml) for the last 2.5 h. Results represent mean ± SD (n = 3 technical replicates) and are representative of two independent experiments. us = unstimulated (a and b). Statistical differences were calculated between the unstimulated condition and the other conditions by Student's t-test adjusted by Bonferroni correction over 6 (a and b). nd, non-detected; ns, non-significant; **p ≤ 0.01; ***p ≤ 0.001.

Discussion
Here we have provided evidence for the immunomodulatory effects of OM-85 on the production of two cytokines playing key roles in lung infections and diseases: type I interferons and IL-1. We found that OM-85 was able to induce IFN-β at an optimal intermediate concentration but less at higher doses. Interestingly, these data are reminiscent of results obtained with other inducers of type I IFNs, including DNA, RNA, and fungal pathogen-derived molecular patterns, which show a bell-shaped dose-response curve [57][58][59][60] . These findings suggested that OM-85-triggered IFN production was compatible with TLR stimulation, a hypothesis that was corroborated by the dependency on both TLR adaptor proteins Trif and MyD88. The ability of OM-85 to induce type I IFN was also observed -to a smaller extent -in human DCs 61 . This represents an important feature potentially involved in the beneficial effects of OM-85 in airway inflammatory diseases, as IFN is a well-known antiviral cytokine, contributing to minimize the occurrence of various respiratory tract infections and the subsequent exacerbations of chronic inflammatory conditions 62,63 .
Whereas OM-85 did not activate the inflammasome per se, it nicely primed BMDCs, significantly rising proIL-1α and proIL-1β levels, in agreement with data in human DCs 61 . Furthermore, our results showed that MyD88 was necessary to augment the levels of proIL-1α and proIL-1β , which are canonical NF-κ B targets, downstream of OM-85. Along these lines, previous findings showed a central role for TLRs, MyD88, and activation of downstream NF-κ B signaling by OM-85 48,51,61 . In addition, we show here the involvement of the adaptors MyD88 and Trif in the induction of type I IFN, inferring the implication of TLR3 or TLR4 upon OM-85 treatment 51 .
In this work we focused on two inflammasome platforms relevant in the context of antiviral responses, and, in particular, of airway infections: AIM2 and NLRP3 16,[23][24][25][26][27] . We showed that OM-85 exposure efficiently pre-activated BMDCs, an effect that was also demonstrated for proIL-1β transcript level on human DCs, for subsequent inflammasome activation and IL-1 production 61 . Given the role of the NLRP3 inflammasome and recent data proving the efficacy of OM-85 in controlling influenza infection, it is conceivable that part of the protective effect of OM-85 is mediated by priming of the inflammasome-IL-1 axis 16,[23][24][25][26][27]56 . Our results indicate therefore that OM-85 treatment keeps innate immune cells in an "alerted state" ideal to release large amounts of IL-1 upon sensing an inflammasome trigger and, conceivably, to reduce selected viral infections.
Yet, the pre-activated state induced by OM-85 in BMDCs is not "inflammatory" per se, as negligible release of IL-1 is measured under these conditions. This is an important aspect when considering the detrimental effects of IL-1 on chronic inflammatory diseases 1 . It therefore seems that OM-85 has the ability to boost inflammation, but exclusively when specific activators of the inflammasome are present. Interestingly, we noticed that the effects of OM-85 on priming, particularly at low doses, diverged in response to different inflammasome activators. In general, activators inducing lower levels of IL-1 release, such as alum, required higher doses of OM-85, indicating a stronger dependency on priming.
Interestingly, we also observed that an overnight pre-treatment with OM-85 dampened the release of IL-1β induced by canonical LPS and alum stimulation. This inhibitory activity was in part due to a reduced activation of caspase-1. These in vitro results were corroborated by in vivo data showing that OM-85 pre-treatment substantially interferes with an IL-1-driven peritonitis model. It is important to stress that in vivo i.p. treatment with alum is sufficient for the secretion of IL-1α and IL-1β, indicating that their precursor proteins are synthesized in response to endogenous priming stimuli. As we showed, OM-85 was per se able to pre-activate BMDCs for inflammasome activation and IL-1 production, suggesting therefore that its immunomodulatory nature renders it activatory in the absence of additional priming signals and -to some extent -inhibitory in their presence. It was however surprising to see that this effect was quite specific to alum and observed upon activation by MSU only when OM-85 was used at very high doses. This indicates that, besides clear differences between soluble and particulate activators 32 , more subtle distinctions exists among the latters, which might reside in their chemical or physical properties, including their size.
Despite a significant body of literature demonstrating that the inflammasome pathway exerts multiple and important functions in the pathophysiology of airway diseases, whether OM-85 regulates such cellular responses remained unanswered. Derived from common human bacterial respiratory tract pathogens and used for the prevention of airway infections and chronic inflammation, OM-85 exhibits the capacity to modulate inflammasome responses. Our data suggest that the mode of action of OM-85 with regard to the IL-1 pathway is twofold. On the one hand, it dampens IL-1β production in response to alum. This effect might be important to prevent selected inflammatory reactions also in the lung, where OM-85 was previously shown to reduce inflammation 48 . On the other hand, OM-85 clearly supports adequate IL-1 production in the case of inflammasome activation. This effect might contribute to the protective effects of OM-85 in the presence of viruses controlled by the inflammasome 56 .

Experimental procedures
Immunoblot analysis. The antibody against mouse IL-1β was a gift from R. Solari (Imperial College, London), whereas the antibody against mouse caspase-1 was a gift from P. Vandenabeele (Ghent University, Belgium) 64,65 . Other antibodies used were monoclonal anti-tubulin from Sigma-Aldrich; rabbit polyclonal antibody to β -actin from Abcam; anti-IL-1α (ALF161) from ebioscience. BMDCs were primed with 10 ng/ml LPS or the indicated doses of OM-85 (referring to μ g/ml) for 4 h, followed by stimulation with alum (300 μ g/ml), MSU (300 μ g/ml), nigericin, (0.1 μ M) for 2.5 h and ATP (500 μ M) for 45 min. Release of IL-1β and IL-1α was measured by ELISA. (c and d) BMDCs were primed as described above, followed by transfection of poly(dA:dT) (0.6 μg). 6 h after, release of IL-1β (c) and IL-1α (d) in the supernatants was measured by ELISA. Results represent mean ± SD (n = 3 technical replicates) and are representative of at least three (a and b) and two (c and d) independent experiments. us = unstimulated. Statistical differences were calculated between the condition exposed to the inflammasome activator only (Alum, MSU, ATP, or Nigericin in the absence of LPS) and the conditions treated with LPS or OM-85 and the inflammasome activator by Student's t-test adjusted by Bonferroni correction over 4 (a and b). Statistical differences were calculated between the condition exposed to poly(dA:dT) in the absence of LPS and the conditions treated with LPS or OM-85 and poly(dA:dT) as well as between the condition treated by LPS and poly(dA:dT) and by OM-85 (1000 μ g/ml ) and poly(dA:dT) using Student's t-test adjusted by Bonferroni correction over 5 (c and d). In vitro stimulation experiments. BMDCs were differentiated as previously described 68 . For normal stimulation experiments, 7 × 10 4 differentiated BMDCs were plated in DC differentiation medium (RPMI 1640, 10% FCS, 100 U/ml penicillin, 100 μ g/ml streptomycin, 50 μN 2-ME, 10 mM HEPES, supplemented with 20 ng/ml rGM-CSF (ImmunoTools)) and treated for the indicated times and with the indicated doses of OM-85 of In vivo peritonitis experiments. For peritonitis, mice were injected i.v., with 2.5 mg OM85 followed 5 h later by an i.p. injection of 350 μ g alum (Pierce). 12-14 h after alum injection, mice were sacrificed and peritoneal cavities were washed with 6 ml PBS. PECs were counted and analyzed by flow cytometry using a combination of antibodies against CD11b (M1/70), Ly6C (AL-21), Ly6G (1A8) (BD Pharmingen), antiCD16/32 (93), CD11c (N418), F4/80 (BM8) (eBioscience). For cellular subtype analysis the following gating strategies were used: neutrophils (CD11b + , Ly6G high , F4/80 − ), eosinophils (CD11b + , SSC high ) and macrophages (Ly6G − , SSC low , CD11b + , F4/80 + ), recruitment was analyzed on a FACSCanto (BD Bioscience) by using the FLOWJO software (Tree Star).
ELISA. Cell culture supernatants were assayed for mouse IL-1β , IL-1α (ebioscience), and IFN-β (PBL Assay Science) according to manufacturer's instructions.  (a-d) C57BL/6 mice were injected intravenously with the indicated dose of OM-85 or PBS as a control. 5 h later, mice were challenged intraperitoneally with 350 μ g of alum. 12 h after alum injection, mice were sacrificed and peritoneal lavages were performed. Absolute numbers of peritoneal exudate cells (PECs) (a), neutrophils (b), eosinophils (c), and macrophages (d) recruited to the peritoneal cavity are depicted. Results represent mean ± SEM (n = 7 mice per group) and are a pool of two experiments and are representative of four independent experiments. Statistical significance between control and OM-85-treated mice was calculated using Student's t-test. **p ≤ 0.01; ***p ≤ 0.001.