Acetate was administered in drinking water after removal of abx treatment. Data are from two independent experiments. The groups were compared using Kruskal—Wallis, except in Fig.
Data in b , c , d , f are provided as a Source Data file. Acetate was added to the drinking water before infection, in a manner similar to the experiments performed with diet Fig. The treatment caused lower weight loss after RSV infection Fig. RSV-F protein was not detected by immunohistochemical analysis in lung tissue of mice pre-treated with acetate, confirming the protective effect of acetate against virus infection Fig.
Propionate and butyrate pre-treatment caused similar protective effects Fig. We also treated mice with acetate in the drinking water simultaneously with intranasal RSV instillation, in order to test for a possible therapeutic effect of acetate Fig. Notably, acetate protected mice against weight loss caused by infection Fig. Acetate started to reduce the viral load significantly at day 3 post infection, culminating in an undetectable viral load in the lung on day 5 Fig. The treatment reduced cell numbers in BALF, including a significant reduction in macrophage and lymphocyte amounts Fig.
Hence, acetate treatment reduced the inflammatory score in the lung Fig. ND not detected. Statistical significance between the groups was determined by Kruskal—Wallis, except in c , in which Mann—Whitney was used to compare the groups. Data in b — d are provided as a Source Data file.
Acetate treatment protects mice against RSV infection. Treatment was performed daily up to 5 days after infection. Data in b , c , d are from two independent experiments. Statistical significance between the groups was determined by Kruskal—Wallis, except in g , h , j , in which Mann—Whitney was used to compare the groups. Data in b , c , d , g , i , j are provided as a Source Data file. Acetate treatment protected against RSV-induced body weight loss Fig.
Acetate treatment protects against RSV infection in the absence of T cells. Data are from two independent. Statistical significance between the groups was determined using Kruskal—Wallis test except in b , in which Mann—Whitney was used. Data in a — d are provided as a Source Data file. We hypothesized that acetate has a direct antiviral effect on pulmonary cells, given its significant effect on diminishing viral loads in the lung.
Propionate and butyrate also protected epithelial lung cells in vitro Supplementary Fig. We then tested the same treatments on Vero cells. The protective effect against RSV-induced cell death was not observed in this cell line Supplementary Fig. There was not a significant increase in the interferon lambda gene expression in A cells treated with acetate Supplementary Fig.
This finding was corroborated by western blot results in which we observed an increase of p65 in nuclear lysate of acetate-treated and -infected cells Fig. Lysis plate titration was performed using an anti-RSV antibody. Panel C shows data co-localization merge. Data in a , f , h , m are quadruplicate mean from 3 experiments.
Data in b , g , l , m are sextuplicate mean from two experiments. Statistical significance was determined with Kruskal—Wallis, except in b , c , g , h , in which Mann—Whitney. We collected fresh lung epithelial cells from naive mice and cultured them.
As demonstrated in Fig. There was no significant difference between the ISGs and interferon lambda expression Supplementary Fig. No significant differences were found in body weight loss Fig.
Moreover, in the absence of the receptor, acetate was unable to cause viral clearance in the lungs Fig. Gate strategy, pre-sorting and post-sorting panel are shown in Supplementary Fig.
Analyses were performed on day 5 post infection. Statistical significance between groups was determined with Kruskal—Wallis, except in e , in which Mann—Whitney was used. Detailed clinical and demographic data have been previously described In our study population, the overall MAF was 0. We hypothesized three potential effects of this functional SNP and tested these with logistic regression models.
We found significant associations in all models tested Supplementary Table 3. In testing for the variant effects, we found the odds ratios were 0. Thus, the variant C Leu is protective against severe disease for those individuals infected with RSV. Incorporation of breastfeeding increased genetic effects in all three models p -values were 0. Given the importance of G-protein coupled receptors i. Considering this, we repeated the in vitro experiments using a potent and selective GPRsynthetic agonist The GPR43 agonist caused the same pattern of response observed with acetate.
It protected the cells against infection Fig. Four days later response parameters were analyzed. Data in a and b are quadruplicate mean of two experiments. Data in c and d are sextuplicate mean of one experiment. Statistical significance was determined with Krukal-wallis. The acetate antiviral effect was also abrogated in these mice Fig. In addition, acetate did not induce Ifnb1 , Oas1 , and isg15 gene expression Fig.
This showed that the acetate treatment protected the mouse epithelial cells from cell death caused by the virus and this effect was abolished in the absence of GPR43 Fig. The same experiment was repeated in GPR43 deficient mice but no differences between treated and control were found Fig. Data in a — g are from two independent experiments.
Statistical significance between the groups was determined with Kruskal—Wallis. Data in a , b , c , f , g , i are provided as a Source Data file. To better understand the potential genetic contribution of Ifnar1 and Gpr43 Ffar2 to the response to RSV, we searched the Jackson Laboratory Mouse Phenome Database for informative SNPs in both the genes to determine whether they associated with RSV-induced phenotypes identified in a previous study of 30 inbred strains of mice We identified a distinct mechanism by which acetate, a microbiota-derived metabolite, impacts immune responses during viral infection in the lung.
Acetate protected against RSV-induced disease by improving type 1 interferon responses and increasing interferon-stimulated gene expression in lung epithelial cells, through a mechanism that involves activation of the membrane receptor GPR These findings highlight the relevance of the gut microbiota and associated metabolites in non-intestinal inflammatory and infectious conditions.
Our data also support the concept of using of high-fiber diets, fiber-enriched formulas or even acetate, as inexpensive interventions for prevention and treatment of bronchiolitis caused by RSV. Lynch et al. In our study, we found a similar prophylactic effect after oral supplementation with propionate, acetate, or butyrate before RSV infection.
This latter result indicates that acetate may be useful in the treatment of RSV infection. In contrast to Lynch et al. This mechanism was relevant for the protection conferred by acetate since we found this effect against RSV infection was abrogated in the absence of type 1 interferon receptor IFNAR in vivo and in vitro.
In addition, the importance of the microbiota for the production of IFN-1 35 , and protection against respiratory viral infection had been demonstrated previously 20 , 36 , The affinity of IFN-beta for the type I receptor is higher than other type 1 interferons being the most potent activator of the receptor. In agreement with a relevant role of IFNAR in the protective effect of acetate, we found that acetate treatment induced the transcription of well-characterized antiviral ISGs, Oas1 encodes oligoadenylate synthetase and Isg15 in the lungs of RSV-infected mice.
The protein encoded by Oas1 has been shown to decrease RSV replication 40 , a mechanism that may explain the acetate effects.
The production of type I interferon during RSV infection must be carefully balanced to reduce viral load without allowing excessive cell infiltration in the lung. We found that acetate modulated the type 1 interferon response on lung epithelial cells, after oral and intranasal treatments.
We hypothesize that this effect of acetate may be associated with a direct effect on epithelial cells that overcome the virus inhibitory mechanisms that act on the IFN-I pathway. Alveolar macrophages are suggested to be the main source of type I interferon and are important to recruit inflammatory monocytes to the lung thus decreasing disease severity However, we did not find that macrophages produced type 1 interferon in response to acetate; the main source of IFN-b in our study was epithelial lung cells.
Macrophages are described to be resistant to RSV replication, not resulting in the production of viral particles. This may lead to a reduced susceptibility in these cells to the effect of RSV NS proteins that are a natural inhibitor of type 1 response Goritzka, M.
Acetate may have a direct effect on epithelial cells that overcome, at least in part, the virus inhibitory mechanisms of RSV NS proteins that act on the IFN-I pathway, and might be the reason why acetate presents its effects in epithelial cells and not in macrophages.
Structural modeling revealed the larger leucine residue provides a stable conformation with better packing in the hydrophobic SD2 cavity Importantly, the IFNAR1 rs mutation is in strong linkage disequilibrium with proximal intronic and distal promoter SNPs associated with viral infection 39 , 48 , We also found that breastfeeding increased the genetic effect of the C allele with protection from severe disease.
Although breastfeeding increased the levels of gut acetate in infants 51 , the involvement of short-chain fatty acids and diet on the genetic protection of IFNAR1 polymorphism is the focus of future studies. In addition to modulation of IFN-I production, acetate treatment reduced the recruitment of inflammatory cells including macrophages and lymphocytes to the lungs of mice, and reduced Th2 and dendritic cells activation in the lymph nodes after 5 days of RSV infection.
These effects might be associated with the well-described pro-resolution effect of acetate 15 and may partially explain the maintenance of the bronchoalveolar organization. ISG15 was associated with IL production 52 , which can also contribute to the anti-inflammatory effect observed in our model. Furthermore, acetate and the other SCFAs, butyrate, and propionate have been described to have a role in activation and function of immune cells 8 , 13 , 53 — Acetate is known to activate GPR43, a receptor that is expressed in the membrane of different cells including epithelial cells This receptor was first characterized in the context of inflammatory conditions such as colitis 9 , in which its activation was shown to attenuate inflammation and to mediate the protective effects of SCFAs in the colon.
Later, the role of this receptor in the context of metabolic and infectious diseases was also demonstrated 57 — Trompette et al. In addition, we found greater susceptibility to RSV-induced disease phenotypes in strains of mice with the loss-of-function SNPs in Ifnar1 and Gpr43 compared to inbred strains of mice with the wild-type alleles. These results are consistent with a protective role for these two genes in the response to RSV infection in mice.
We found that HF-diet-fed mice had an increase of gut bacterial components of Lachnospiraceae spp, which are in a bacterial family usually isolated from human intestinal microbiota, including from infants in the first year of life A study showed a decrease in the Lachnospiraceae family following RSV infection in mice Increased abundance of this family has also been reported in other studies in which fermentable fiber was added, and was associated with increased serum levels of acetate This mechanism might be also relevant for protection against other virus infection.
All animals were weighed daily. Data analysis was performed 5 days post infection or as indicated. Bronchoalveolar lavage fluid BALF was collected and left lungs were removed after perfusion with formalin for Histopathological and Immunohistochemistry analysis.
Mice were fed with high-fiber diet HF or control diet CF for 4 weeks before infection. In another experiment, high-fiber diet-fed mice received an antibiotic mix consisting of kanamycin 0. All antibiotics were purchased from Sigma—Aldrich. Another SCFA treatment was performed starting simultaneously with the infection at the same final concentration.
Water volume consumption was measured daily and no difference in water intake was found between the groups. Mice were anesthetized with intraperitoneal administration of ketamine 0. The lungs were washed twice with RPMI medium.
BALF was centrifuged, the supernatant collected for cytokine analysis and pellets suspended for total cell and differential count and flow cytometry. Slide analysis was performed in a blinded manner. The peribronchial and perivascular inflammation was scored according to Barends et al. The binding capacity of the oligosaccharides has proven protective against viruses with high morbidity and mortality, including human immunodeficiency virus HIV 55 and rotavirus 56 , and breast milk mucin is able to aggregate poxviruses prior to their entry into host cells Additional protective properties of breast milk are provided by the transfer of maternal immune cells to the infant, including macrophages, neutrophils and lymphocytes The concentration of these cells in the human breast milk vary according to the age of the infant, taking into account that in the early stage of lactation, the neonatal immune system is completely immature The immune properties of the mother are also transferred to the breastfed infant in the form of secretory IgG 60 and IgA in maternal milk Breast milk attains the highest concentration in IgG antibodies in the colostrum, and their concentration drops after the first month of life 62 and stops abruptly with weaning In mothers immunized against RSV, s-IgG antibodies were detected in breastmilk 64 , providing protection to the infant against the main cause of respiratory infection during the first year of life IgA antibodies coat the GI and respiratory mucosa and block the entrance of foreign antigens 50 and viruses In premature infants, the IgA levels are higher, for enhanced protection In the event of an infection, in either the mother or the child 67 , breast milk conveys a plethora of antipathogenic and anti-inflammatory bioactive factors 68 to protect the infant.
In attacks by respiratory virus infections, such as RSV, protection is mediated by polymeric IgA antibodies to a protein of the RSV surface membrane, inhibiting virus replication In addition to the transfer of antibodies, human milk triggers immune-protective responses by the host. In influenza, type I interferons IFN , cytokines with strong simultaneous anti-viral, pro-apoptotic and pro-inflammatory properties, are produced significantly more often in breastfed infants, transforming viral attacks to formes frustes Bacteria in the human milk are one of the earliest sources of prokaryotic microorganisms transferred to the infant, following the maternal microbial colonization through the amniotic fluid, placenta and umbilical fluid 72 , and a more substantial transmission of vaginal and gut microorganisms to the newborn through the birthing canal HMM and other human milk components, such as human milk oligosaccharides HMO , reflect environmental factors, e.
Human milk transmits a significant load of viruses, eukaryotic and bacteriophages, from the mother to the infant, which enhance the maturation of both innate and adaptive immune systems. Eukaryotic viruses may impact the health status directly, while bacteriophages act via the bacterial ecology.
In children, COVID infection rates are lower than in adults, while fatality rates are almost zero Children generally have milder symptoms, but there are reports of the development of a novel multisystem inflammatory syndrome in children MIS-C similar to Kawasaki disease, predicating continuous vigilance The current policy is that breastfeeding is contraindicated in only a limited number of viral diseases, i.
The unsurpassed benefits of breastfeeding include not only irreplaceable nutrition, ensuring healthy growth for the infant, but also prevention of obesity in later life It is a mainstay for promoting the immune development of the infant by both immunological factors transferred from mother to infant through breast milk 76 and microorganisms colonizing the organs and viruses enriching the virome. SARS-CoV-2 is probably transmitted in multiple ways, including through droplets via the respiratory tract and invasion by enterocytes GI symptoms manifest first in infancy, and lactoferrin in breast milk has been suggested to be capable of strengthening junctions between microbes in the gut and thus amplifying the innate defense.
The most abundant antibody in breast milk, sIgA, provides adequate specific protection against pathogens, among which also are viruses. The specificity of sIgA is determined by the immune response of the mother to previous infection, probably explaining the low rates of infection or milder symptoms of the infected breastfed infants of SARS-CoV—infected mothers.
Concerning this phenomenon, the cases where infection is not protected against by breastfeeding should be considered. For example, in pertussis, the sIgA in the human milk of infected mothers was higher than in that of control subjects, but did not protect against infection of the infant, and therefore vaccination of pregnant women was recommended The evidence to date on pregnant women infected by SARS-CoV-2 does not demonstrate a more severe or complex clinical picture than in the general infected population.
In view of the time-dependent protective effect of exclusive breastfeeding against viral infections, and the increased maternal contact of the breastfed infant compared with the infant receiving artificial or mixed feeding 41 , and the high transmissibility of SARS-CoV, protective measures should be strictly observed for safeguarding the lactation process.
EV contributed to the conception, designed the review, and wrote the first draft. EV and GF collected the relevant literature and independently reviewed the studies, while GK further evaluated the studies where conflict was presented in the decision. All authors contributed to the article and approved the submitted version. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Emerg Microbes Infect 9 1 —9. J Infect 81 3 — Lack of vertical transmission of severe acute respiratory syndrome Coronavirus 2, China. Emerg Infect Dis 26 6 —6.
Lancet —8. Clin Infect Dis —6. Breastfeed Med 15 5 —8. A case report of neonatal coronavirus disease infection in South India.
Indian J Case Rep 6 8 —3. Probable congenital sars-cov-2 infection in a neonate born to a woman with active sars-cov-2 infection. Cmaj 24 :E— Int J Infect Dis —4. A case report of neonatal coronavirus disease in China. Clin Infect Dis 71 15 —7. Clin Infect Dis —8. Saleh Elhalik M. No effect on IFN production was observed for age, gender or smoking.
Conclusion: Our study confirms that type I IFN production is detected more frequently in infants infected with influenza virus. Importantly, higher rates and levels of type I IFN in these infants are associated with breastfeeding. These observations suggest that breast milk can protect against respiratory viruses by activating innate antiviral mechanisms in the host.
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