USE OF SHORT-CHAIN FATTY ACIDS FOR THE TREATMENT OF BACTERIAL SUPERINFECTIONS POST-INFLUENZA

Abstract

Severe influenza is associated with defects in pulmonary innate immunity, a phenomenon leading to secondary bacterial infections. The gut microbiota can control immune/inflammatory responses locally and at distant sites. The inventors hypothesized that perturbation of the gut microbiota during severe influenza might participate in bacterial superinfection post-influenza. Their data demonstrated that influenza infection profoundly altered the functionality of the gut microbiota as assessed by the altered production of short chain fatty acids (SCFAs). Remarkably, treatment of colonized (IAV microbiota) mice or IAV-infected mice with acetate, the main SCFA found systematically, reinforced host defenses against S. pneumoniae. The present invention thus relates to the use of short-chain fatty acids for the treatment of bacterial superinfections post-influenza.

Claims

1. A method of treating a bacterial superinfection post-influenza in a subject in need thereof comprising administering to the subject a therapeutically effective amount of at least one short-chain fatty acid (SCFA) after an influenza infection.

2. The method of claim 1 wherein the at least one SCFA is selected from free fatty acid receptor 2 (FFAR2) agonist or free fatty acid receptor 3 (FFAR3) agonist.

3. The method of claim 1 wherein the influenza infection is caused by Influenza virus A or B.

4. The method of claim 3 wherein the influenza virus A is H1N1, H2N2, H3N2 or H5N1.

5. The method of claim 1 wherein the bacterial superinfection is selected from the group consisting of lower respiratory tract infections, middle ear infections and bacterial sinusitis.

6. The method of claim 1 wherein the bacterial superinfection is caused by at least one organism selected from the group consisting of Streptococcus pneumoniae; Staphylococcus aureus; Haemophilus influenza, Myoplasma species and Moraxella catarrhalis.

7. The method of claim 1 wherein the subject is selected from the group consisting of subjects who are at least 50 years old, subjects who reside in chronic care facilities, subjects who have chronic disorders of the pulmonary or cardiovascular system, subjects who required regular medical follow-up or hospitalization during the preceding year because of chronic metabolic diseases, renal dysfunction, hemoglobinopathies, or immunosuppression, children less than 14 years of age, patients between 6 months and 18 years of age who are receiving long-term aspirin therapy, and women who will be in the second or third trimester of pregnancy during the influenza season.

8. The method of claim 1 wherein the subject is older than 1 year old and less than 14 years old; between the ages of 50 and 65, or older than 65 years of age.

9. The method of claim 1 wherein the at least one SCFA is selected from saturated fatty acids comprising six or less carbon atoms, or 5 or less carbon atoms.

10. The method of claim 1 wherein the at least one SCFA is selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, formate, acetate, butyrate, isobutyrate, valerate, isovalerate, caproate, propionic acid and propionate.

11. The method of claim 1 wherein the SCFA is administered to the subject in the form of a nutritional composition.

12. The method of claim 1 wherein the SCFA is administered to the subject in the form of a food composition.

13. The method of claim 1 wherein the SCFA is administered to the subject in a pharmaceutical composition.

14. A method for determining whether a subject suffering from an influenza infection is at risk of having a bacterial superinfection and treating the subject, comprising i) determining the amount of short-chain fatty acid in a sample obtained from the subject, ii) comparing the amount determined at step i) with a predetermined reference value and iii) administering a at least one short chain fatty acid to the subject when the amount determined at step i) is lower than the predetermined reference value.

15. The method of claim 14 wherein the sample is a fecal sample or a blood sample.

Description

FIGURES

[0039] FIG. 1. Protective effect of acetate treatment on the control of pneumococcal infection. (A) Mice were treated or not with acetate five days before with S. pneumoniae (110.sup.6 CFUs) challenge. The number of bacteria was determined in lungs (upper panel) and spleen (lower panel) 30 hours after S. pneumoniae challenge. The solid lines correspond to the median values. A pool of two experiments is shown (n=18). (B) The mean number of bacteria+/SD was determined in acetate-treated wild type (WT) and Ffar2.sup./ mice. A representative experiment is shown (n=5). (A, B), **P<0.01, *P<0.05 (in a Mann-Whitney U test).

[0040] FIG. 2. Reduced metabolic activity of the gut microbiota during influenza infection. (A), Cecal concentrations of total SCFAs in mock-infected mice and 7 and 14 days post-IAV (H3N2) infection (left panel). The concentration of the main SCFAs in gut microbiota is depicted in the right panel (n=24, three pooled experiments). (B), Cecal concentrations of total SCFAs during the course of IAV (H1N1) infection (n=8). Data are expressed as meanSD. Significant differences were determined using a Kruskal-Wallis one-way ANOVA test (A) or a Mann-Whitney U test (B) (***, P<0.001).

[0041] FIG. 3. Enhanced susceptibility to respiratory bacterial infection of mice colonized with IAV-experienced microbiota. The microbiota from mock-infected (control) mice or from IAV-infected (7 dpi) mice (A, H3N2, B, H1N1) was transplanted to ABX-treated mice. Three days later, colonized mice were challenged with S. pneumoniae (110.sup.6 CFUs). Bacterial loads were determined 30 hrs later. The solid lines correspond to the median values. A pool of two experiments is shown. *P<0.05, **, P<0.01) (in a Mann-Whitney U test).

[0042] FIG. 4. Protective effect of acetate treatment on the control of bacterial infection in colonized (IAV-experienced microbiota) mice and in IAV-infected mice. (A) The microbiota from IAV-infected (7 dpi, H1N1) mice was transplanted to ABX-treated mice and three days later, colonized mice were challenged with S. pneumoniae (110.sup.6 CFUs). Colonized mice were treated or not with acetate five days before S. pneumoniae challenge. (B) IAV (H1N1, WSN/33)-infected mice were treated with acetate at day 2 post-infection and were challenged with S. pneumoniae (110.sup.3 CFUs) at 7 dpi. (A and B), CFUS were determined (lungs and spleen) 30 hrs later. The solid lines correspond to the median values. A pool of two experiments is shown. *P<0.05, **, P<0.01 (in a Mann-Whitney U test).

[0043] FIG. 5. Protective effect of acetate treatment on the control of bacterial infection in IAV-infected mice. IAV (H1N1, A/California/04/2009)-infected mice were treated with acetate at day 2 post-infection and were challenged with S. pneumoniae (110.sup.3 CFUs) at 7 dpi. (A), CFUS were determined in the lungs (upper panel) and in the spleen (lower panel) 30 hrs later. The solid lines correspond to the median values. A representative experiment out of two is shown (n=8-9) (Mann-Whitney U test). (B). The survival of superinfected animals was monitored (n=14/group, two pooled experiments) (Kaplan-Meier analysis and log-rank test). *P<0.05, **, P<0.01.

EXAMPLE

[0044] Methods

[0045] Mice, Ethics Statement and Reagents

[0046] Specific pathogen-free C57BL/6 mice (6-8 week-old, male) were purchased from Janvier (Le Genest-St-Isle, France). Mice were maintained in a biosafety level 2 facility in the Animal Resource Center at the Lille Pasteur Institute. All animal work conformed to the Lille Pasteur Institute animal care and used ethical guidelines (agreement number AF 16/20090 and 00357.03). Antibiotics were from Sigma-Aldrich (St Louis, Mo.) or R&D systems (Minneapolis, Minn.). SCFAs were from Sigma-Aldrich. FFAR2.sup./ were described in (Maslowski et al., 2009).

[0047] Infections and Assessment of Bacterial Load

[0048] Mice were intranasally (i.n., 501) infected with the high-pathogenicity mouse-adapted H3N2 IAV strain Scotland/20/74 (30 plaque forming units, PFUs), H1N1 IAV strain WSN/33 (200 PFUs) or H1N1 IAV strain A/California/04/2009) (100 PFUs) (Barthelemy et al., 2016; Barthelemy et al., 2017, Barthelemy et al. 2018). In the case of single bacterial infection, mice were i.n. inoculated with 110.sup.6 colony-forming units (CFUs) of S. pneumoniae serotype 1, a serotype linked to invasive pneumococcal disease (clinical isolate E1586). Superinfection was as follows. Mice were infected with IAV (the sub-lethale doses indicated above) and seven days later, animals were i.n. inoculated with 110.sup.3 CFUs of S. pneumoniae). A high severity model (prior infection with IAV H1N1, A/WSN/1933) and a mild severity model (prior infection with IAV H1N1, A/California/04/2009) of superinfection were used in this study. Enumeration of viable bacteria in lungs and spleen was determined 30 hours after the S. pneumoniae challenge. Survival was monitored daily after IAV infection and mice were euthanized when they lost in excess of 20% of their initial body weight.

[0049] Measurement of SCFA Concentrations and SCFA Treatment.

[0050] Concentrations of SCFAs in the cecal contents were determined using high-performance liquid chromatography by the internal standard method (LC-6A; Shimadzu, Kyoto, Japan) equipped with a Shim-pack SCR-102H column (inner diameter, 8 mm; length, 30 cm; Shimadzu) and a CDD-6A electroconductivity detector (Shimadzu). Nave (non-infected) mice and IAV-infected mice were treated with acetate (200 mM, drinking water) five days before S. pneumoniae challenge (110.sup.6 CFUs and 110.sup.3 CFUs, respectively).

[0051] Fecal Transfer Experiments

[0052] Mice received broad-spectrum (fresh) antibiotics (ampicillin 2 g/L; neomycin 2 g/L, metronidazole 1 g/L, cyproflaxyn 1 g/L, nystatin 0.08 g/L and vancomycin 0.5 g/L) in drinking water for three weeks. The cages were changed every two days. Depletion of bacteria in the feces were checked by plating experiments. Antibiotic-treated mice were colonized (three days after antibiotics cessation) with 110.sup.9 bacteria recovered from mock-infected mice or from IAV-infected mice (7 dpi). The procedure was repeated two days after. One day after the last colonization, mice were infected with S. pneumoniae (110.sup.6 CFUs).

[0053] Statistical Analyses

[0054] A Mann-Whitney U test was used to compare two groups unless otherwise specified. Comparisons of more than two groups with each other were analyzed with the One-way Anova Kruskal-Wallis test (nonparametric), followed by the Dunn's posttest (PRISM v6 software, GraphPad Results are expressed as the meanstandard deviation (SD) unless otherwise stated. Survival of mice was compared using Kaplan-Meier analysis and log-rank test. A value of P<0.05 was considered as significant.

[0055] Results

[0056] Treatment with Acetate Protects Against Pneumococcal Infection

[0057] Short chain fatty acids are amongst the most abundant molecules produced by the gut bacteria. Production of gut microbiota-derived SCFAs has recently been shown to modulate pulmonary immune responses (asthma reaction), although the consequences on respiratory infections is still elusive (Maslowsky et al., 2009; Thorburn et al., 2015; Trompette et al., 2014; Cait et al., 2017). To investigate the effect of SCFAs on host defense against S. pneumoniae, mice were treated during five days with acetate (drinking water), the major specie of SCFAs. Compared to vehicle-treated animals, acetate supplementation significantly reduced the number of bacteria in the lungs (FIG. 1A, upper panel). Streptococcus pneumoniae can disseminate out of the lungs to become invasive. Of interest, acetate supplementation also lowered the number of bacteria in the spleen (FIG. 1A, lower panel). Hence, acetate boosts lung immunity to control pneumococcal infection. Acetate predominantly acts through the G-coupled receptor FFAR2 (also known as GPR43) and, to a lower extent (at least in humans), to FFA3 (GPR41) (Milligan G et al., 2017). As depicted in Figure. 1B, and relative to FFA2-competent mice, the protective effect of acetate was significantly reduced in Ffa2.sup./ mice.

[0058] Influenza Infection Alters the Fermentation Activity of the Gut Microbiota

[0059] We next questioned whether a drop of SCFA production during severe influenza could lower pulmonary host defense against pneumococcal infection. Before this, we measured SCFA production in the context of influenza infection. As shown in FIG. 2A (left panel), the concentration of total SCFAs was significantly diminished 7 days post-influenza (H3N2) infection to return to basal level at 14 dpi. The concentration of acetate (C2), propionate (C3) and butyrate (C4), was significantly decreased at 7 dpi (FIG. 2A, right panel). Altered production of SCFAs was also observed in mice infected with H1N1 IAV (FIG. 2B and not shown). Together, this suggests that severe influenza alters the metabolic activity of the gut microbiota.

[0060] IAV-Experienced Microbiota Confers Susceptibility to Respiratory Bacterial Infection

[0061] We next investigated whether IAV-experienced microbiota (through SCFA production) could confer susceptibility to respiratory bacterial infection. To this end, fecal transfer experiments were performed in mice previously treated with antibiotics, a procedure that transiently depletes host commensals. The microbiota collected from mock-infected (control) mice and from IAV (H3N2)-infected mice (7 dpi) was transplanted to ABX-treated mice. Remarkably, and compared to the control group, the transfer of IAV-experienced microbiota enhanced susceptibility to pneumococcal infection (FIG. 3A). To investigate whether this is also the case during H1N1 infection, the same procedure was repeated but this time with the microbiota collected from IAV (H1N1)-infected mice. As depicted in FIG. 3B, mice colonized with IAV-experienced microbiota had a higher number of bacteria in the lungs. In both systems (H3N2 and H1N1), IAV microbiota also enhanced bacterial dissemination out of the lungs (FIG. 3C and not shown). These data suggest that disruption of the intestinal bacterial homeostasis during influenza infection is sufficient to confer susceptibility to respiratory bacterial infection.

[0062] Reduced SCFA Production by IAV-Experienced Microbiota Enhances Secondary Bacterial Infection

[0063] We next questioned whether acetate could rescue the altered response in colonized (acetate-deficient) mice. Remarkably, mice colonized with IAV microbiota and treated with acetate were more resistant to S. pneumoniae challenge compared to vehicle-treated transplanted animals (FIG. 4A). Indeed acetate-treated mice had a lower number of pneumococci in lungs. Acetate treatment also lowered the systemic dissemination of pneumococci out of the lungs. These data suggested that defective SCFA (acetate) production during severe influenza might enhance secondary bacterial infection. To further demonstrate this, IAV-infected mice were treated with acetate before the secondary bacterial challenge. To this end, a severe (H3N2) and a milder (H1N1p) models were developed. In IAV (H3N2)-infected mice supplemented with acetate (at day 2 p.i.), a significant lower bacterial load in the lung and spleen was observed (FIG. 4B). The effect of acetate in the less severe superinfection model was next assessed. Acetate treatment significantly reduced the bacterial loads in the lung and spleen (FIG. 5A). Lastly, and importantly, acetate treatment resulted in a significant enhancement of the survival rate of double-infected mice (FIG. 5B). Taken as a whole, low SCFA production by the gut microbiota during influenza infection influences susceptibility to secondary bacterial infection and restoration of acetate is sufficient to improve disease outcomes.

[0064] Discussion:

[0065] The present study aimed at analyzing the impact of severe influenza on the functionality (fermentation activity) of the gut microbiota and at studying the impact of potential alterations on secondary respiratory bacterial infection, a phenomenon that arises following local immune suppression. This study demonstrates the impact of microbiota alterations during influenza infection on secondary bacterial infection. It also highlights the importance of gut microbiota-derived SCFAs in pulmonary innate immunity against bacterial infections, including in the context of prior influenza.

[0066] Emerging evidences suggest that natural (humans, H7N9) and experimental (mouse, H1N1) influenza infection alters the composition of the gut microbiota (Wang et al., 2014; Lu e al., 2014; Qin et al., 2015; Deriu et al., 2016; Bartley et al., 2017). We have recently analyzed, on a large number of mice, the impact of severe H3N2 and H1N1 influenzathe two mains subtypes in humans-on the diversity and composition of the gut (caecal and colonic) microbiota. We have confirmed that severe influenza alter the relative abundances of microbial taxa at 7 dpi (manuscript in preparation). In contrast to chronic pathologies where a strong decrease of phylogenic diversity arises (dysbiosis), marked changes occurred on phylogenetic specifications, with no major decreased global diversity. We did uncover several shared responses of the microbiota between H3N2 and H1N1. In the current study, we show for the first time that alteration of the gut microbiota composition at 7 dpi is associated with a concurrent drop in the concentration of intestinal SCFAs, an effect probably due to reduced frequencies of bacterial SCFA producers. Among candidates (reduced frequencies at 7 dpi) are S24-7 family (Bacteroidetes) and Lachnospiraceae genus (Firmicutes) which are notable for containing many species capable of fermenting complex carbohydrates to SCFAs (Louis et Flint, 2017; Vital et al., 2014).

[0067] What are the consequences of altered microbiota on disease outcomes during influenza? Deriu and collaborators elegantly demonstrated that gut disorders during influenza favors local colonization and systemic dissemination of Salmonella Typhimurium, a leading cause of acute gastroenteritis (Deriu et al., 2016). The mechanisms leading to this enhanced secondary enteric susceptibility are still elusive and might result from a relaxed intestinal barrier and/or local immune suppression. Whether the drop of SCFA production during influenza plays a role in this setting is an open question. Whatever the mechanisms, this datum suggests that secondary (systemic) infection during severe influenza might originate from the gut compartment, and not solely from the pulmonary compartment. Whether altered gut microbiota might predispose to secondary pulmonary bacterial infection was addressed for the first time in the current study. This is a particularly significant question in view of reported cases of fatal respiratory superinfection during severe influenza. In the present study, we demonstrated that gut microbiota (metabolic) changes have implication in secondary respiratory bacterial infection post-influenza. Indeed, transfer experiments indicate that the altered microbiota affects the pulmonary host response to S. pneumoniae. Acetate is the likely mediator of this defective host response as acetate (the main SCFA distributed throughout the body) in drinking water rescued host defense mechanisms in our settings. To the best of our knowledge, this is the first time that altered SCFA production during a stressful condition leads to impaired immunity and secondary infection.

[0068] The role of acetate (and more generally bacterial metabolites such as SCFAs) in pulmonary host defenses is largely elusive. Short-chain fatty acids are derived from the anaerobic fermentation of non-digestible polysaccharides, such as resistant starches and dietary fibers. Microbial-derived SCFAs exert many physiologic functions in the intestine where their concentration in the gut lumen can reach 100-200 mM (Thorburn et al., 2014). They are (primarily butyrate) a source for host colonic epithelium and enhance intestinal barrier properties. SCFAs display anti-oxidative and anti-inflammatory functions, thus protecting against tumor growth and colitis (Maslowski et al., 2009; Kim et al., 2013). Production of SCFAs is important in the control of the gut microbiota as they can act as energy source for certain bacterial species. Intestinal SCFAs can also protect against enteric infection including shigellosis and salmonellosis (Rabbani et al., 1999; Raqib et al., 2006; Canani et al., 2011; Raqui et al., 2012; Sunkara et al., 2012). SCFAs can diffuse in the blood (0.1-1 mM) and activate numerous physiologic processes. They are very important in the so-called gut-brain axis and in the stimulation of neural and hormonal signals regulating energy homeostasis (Kuwahara, 2014). Recent evidences suggest that SCFAs can act in the lung compartment to modulate pulmonary immunity. For instance, Trompette and coauthors showed that propionate, by acting on dendritic cell progenitors in the bone marrow, lowers Th2 response to allergens, thus decreasing asthma reactions (Trompette et al., 2014). On the other hand, butyrate supplementation alters the function of systemic dendritic cells to ameliorate asthma (Cait et al., 2017). More recently, it was shown that butyrate controls influenza infection by reducing, through enhanced CD8.sup.+ T cell activity, the viral replication (Trompette et al. 2018). SCFAs act though G-protein coupled receptors and/or through HDAC inhibition. The potential function (through supplementation) of SCFAs in respiratory bacterial infections has recently been reported. Interestingly, combined butyrate and vitamin D3 treatment ameliorates pulmonary tuberculosis in humans, probably by boosting antibacterial functions of macrophages (Mil.sub.t et al., 2015). On the other hand, Ciarlo and coauthors show that propionate failed to modulate host defenses against respiratory bacterial (including pneumococcal) and fungal infections (Ciarlo et al., 2016). Our data contrast with this later study, a finding that can be explained by the use of acetate in the current study and the different protocols used for infection. Our data suggests that the protective effect of acetate during S. pneumoniae infection depends on FFAR2 expression. Of importance, treatment of IAV-infected mice with acetate results in a better control of bacterial growth in the lungs and dissemination out of the lungs (both in the severe and mild models of surinfection). In the later system, acetate treatment also resulted in enhanced survival rate. Immunological mechanisms sustaining these protective effects are still elusive and may be due to enhanced number and/or functions of effector cells (macrophages, neutrophils) in the lungs. Cells involved in their activation, including dendritic cells and non-conventional T cells ( and NKT cells for instance) may also be impacted. Whatever the mechanisms, our data highlight the positive role of SCFA supplementation (used in the clinics) during influenza. This finding might have therapeutic applications in many settings including in acute diseases associated with altered microbiota and secondary infections such as trauma, burn and sepsis. To conclude, we propose that altered gut microbiota during acute infection (influenza) acts as a critical factor in secondary respiratory bacterial infection through suppression of host pulmonary immunity. Our findings might have therapeutic applications (SCFAs or FFAR agonists) in diseases associated with dysbiosis and secondary bacterial infections.

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