High body temperature increases gut microbiota-dependent host resistance to influenza A virus and SARS-CoV-2 infection

doi:10.21203/rs.3.rs-1737468/v1

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High body temperature increases gut microbiota-dependent host resistance to influenza A virus and SARS-CoV-2 infection

https://doi.org/10.21203/rs.3.rs-1737468/v1

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While a common symptom of influenza and coronavirus disease 2019 (COVID-19) is fever, its physiological role on host resistance to viral infection remains less clear. Here, we demonstrate that exposure of mice to the high ambient temperature of 36 °C increase host resistance to viral pathogens including influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). High heat-exposed mice increase basal body temperature over 38 °C to enable more bile acids production in a gut microbiota-dependent manner. The gut microbiota-derived deoxycholic acid (DCA) and its plasma membrane-bound receptor Takeda G-protein-coupled receptor 5 (TGR5) signaling increase host resistance to influenza virus infection by suppressing virus replication and neutrophil-dependent tissue damage. Furthermore, the DCA and its nuclear farnesoid X receptor (FXR) agonist protect Syrian hamster from lethal SARS-CoV-2 infection. Moreover, we demonstrate that certain bile acids are reduced in the plasma of COVID-19 patients who developed moderate I/II disease compared with minor illness group. These findings uncover an unexpected mechanism by which virus-induced high fever increases host resistance to influenza virus and SARS-CoV-2 in a gut microbiota-dependent manner.

Respiratory infectious diseases, such as influenza and coronavirus disease 2019 (COVID-19), cause substantial morbidity and mortality. These infectious diseases mostly affect older adults^1,2. Increased susceptibility to influenza virus infection in older humans could be explained by the fact that older human monocytes have impaired signaling to induce type I interferons (IFNs) in response to influenza virus infection^3,4. However, the role of other age-related changes of host factors in susceptibility to influenza virus infection remains unclear. With respect to age-related changes of host factors, several studies demonstrated that the composition of gut microbiota changes with age in both humans and animals^5-7. In addition, mean body temperature in humans decrease with age⁸. Although it has become increasingly evident that gut microbiota and their metabolites are important for protection against influenza virus infection^9-12, the effects of core body temperature on host defense to influenza virus infection are largely unknown.

In addition to age-related changes of host factors, environmental parameters and eating behavior could affect host susceptibility to influenza virus infection^13-15. A recent study showed that mice housed in 10 or 20% relative humidity (RH) succumbed to influenza virus infection more rapidly than those housed in 50% RH¹³. Further, ketogenic diet-fed mice had improved survival relative to mice on a normal chow diet after influenza virus challenge¹⁴. In vitro studies using rhinovirus and influenza virus revealed that warm temperature restricts viral replication through type I IFN-dependent and -independent mechanisms^16-18. In addition, both humidity and temperature affect the frequency of influenza virus transmission among guinea pigs^19,20. We previously demonstrated that exposure of mice to the high ambient temperature of 36 °C impairs virus-specific CD8⁺ T cell responses and antibody production after intranasal challenge with a sublethal dose (30 pfu) of influenza virus¹¹. In contrast, the effects of outside temperature on host resistance to lethal influenza virus challenge remain unknown. Thus, here we explore the effects of outside and core body temperature on host resistance to influenza virus infection.

In this study, we demonstrate that exposure of mice to the high ambient temperature of 36 °C increases body temperature and host resistance to influenza virus or SARS-CoV-2 infection. Moreover, we demonstrate that high body temperature-dependent activation of gut microbiota increases the levels of bile acids in the serum and intestine, and suppresses virus replication and detrimental inflammatory responses following influenza virus and SARS-CoV-2 infection.

Temperature affects severity of influenza

In mice infected with influenza virus, reduction of core body temperature became apparent starting around day 4 p.i. (Extended Data Fig. 1a). Consistent with the reduction of body temperature, the virus-infected mice remained huddled together probably to avoid reducing their body temperature (Extended Data Fig. 1b). These observations prompted us to explore the functional role of core body temperature in resistance to influenza virus infection. To this end, we kept mice at 4, 22, or 36 °C for 7 days before influenza virus infection¹¹. Cold-exposed naïve mice exhibited significant decrease in body temperature compared with room temperature (RT)-exposed group, whereas high-heat exposure of naïve mice significantly increased body temperatures ranged from 38.4 °C to 39.1 °C (Fig. 1a). To examine the role of core body temperature in protection against influenza virus infection, cold-, RT-, or high heat-exposed mice were infected intranasally with a mouse-adapted influenza A virus strain A/Puerto Rico/8/1934 (PR8) and kept at 4, 22, or 36 °C, respectively, for the entire duration of the experiments. After influenza virus infection, cold-exposed mice succumbed to disease with severe hypothermia faster than RT-exposed group, whereas high heat-exposed mice were highly resistant to influenza virus infection at three challenge doses (Fig. 1b, c and Extended Data Fig. 2). Consistent with our previous report¹¹, the virus titers were significantly reduced in the lung of high heat-exposed mice compared with cold- or RT-exposed groups at early time points (Fig. 1d). Similar results were observed when mice were challenged with other viral pathogens such as a human isolate of the 2009 pandemic influenza A virus strain A/Narita/1/2009 (pdm09) (Extended Data Fig. 3), SARS-CoV-2 (Extended Data Fig. 4 and 5), severe fever with thrombocytopenia syndrome phlebovirus (Extended Data Fig. 6), Zika virus (Extended Data Fig. 7), and encephalomyocarditis virus (Extended Data Fig. 8). In addition, aged mice had lower body temperature than adult mice and were more susceptible to influenza virus infection as reported previously (Fig. 1e, f)^21,22, whereas high-heat exposure of the aged mice had improved survival relative to control mice (Extended Data Fig. 9). Further, decrease in basal body temperature of RT-exposed adult mice by partial hepatectomy exacerbated influenza virus-induced mortality (Fig. 1g, h). Together, these data indicated that core body temperature significantly affects host resistance to a broad range of viral infections including influenza A virus and SARS-CoV-2.

High fever increases gut microbial metabolism

Next, we wished to determine the threshold of core body temperature required for protection against influenza virus infection. To this end, we kept mice at 22, 28, 34, or 36 °C before influenza virus infection. While most high heat-exposed naïve mice kept at 36 °C had basal body temperature over 38 °C, an average of basal body temperature of naïve mice kept at 34 °C was 37.2 °C ranged from 37.0 °C to 37.7 °C (Fig. 2a). Although all mice kept at 36 °C survived from lethal influenza virus infection, the majority of the virus-infected mice kept at below 34 °C succumbed to disease at two challenge doses (Fig. 2b and Extended Data Fig. 10a). In addition, all 36 °C-exposed mice kept their body temperature over 38 °C during influenza virus infection (Fig. 2c and Extended Data Fig. 10b). Further, the virus titers were significantly reduced in the lung of high heat-exposed group compared with 34 °C-exposed group at 2 and 3 d p.i. (Fig. 2d). These data indicated that high body temperature over 38 °C may increase host resistance to influenza virus infection.

Recent studies have demonstrated that gut microbiota-derived metabolites protect from influenza virus-induced mortality by enhancing type I IFN signaling or attenuating the neutrophil influx into the lung^9,23. Thus, we hypothesized that gut microbial activity may be activated by host body temperature over 38 °C^24,25. To test whether gut microbiota or microbial metabolites are needed to increase host resistance to influenza virus infection in high heat-exposed mice, we infected high heat-exposed low fiber (LF)-fed or antibiotics (Abx)-treated mice with influenza virus. Strikingly, high heat-exposed LF-fed or Abx-treated mice succumbed to disease despite having body temperature over 38 °C similar to high heat-exposed control mice during influenza virus infection (Fig. 2e, f). Recently, it has been reported that Syrian hamsters are a useful small animal model for SARS-CoV-2 infection²⁶. We found that Abx-treated naïve hamsters kept at 22 °C exhibited significant decrease in basal body temperature compared with control or LF-fed naïve hamsters (Extended Data Fig. 11a). Consistent with a previous report²⁶, control hamsters kept at 22 °C were resistant to infection with an original SARS-CoV-2 strain bearing aspartic acid at position 614 of spike (S) protein (S-614D) (Extended Data Fig. 11b). In contrast, LF-fed and Abx-treated hamsters succumbed to infection despite having viral loads similar to control hamsters (Extended Data Fig. 11b-d). These data indicated that inability of high heat-exposed LF-fed and Abx-treated mice or RT-exposed LF-fed and Abx-treated hamsters to protect against lethal influenza virus or SARS-CoV-2 challenge was not due to their body temperature but may be instead due to reduction of gut microbial fermentation products.

To gain insight into the mechanisms underlying gut microbiota-derived metabolites mediated host resistance to influenza virus infection in high heat-exposed mice, we performed capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), gas chromatography–mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolome analysis of cecal contents, serum, and livers of naïve mice kept at 4, 22, or 36 °C for 7 days. The CE-TOFMS-based cecal metabolome profiles were clustered and found that the level of propionate and both primary and secondary bile acids were significantly increased in the high heat-exposed mice or hamsters compared with cold- or RT-exposed groups (Extended Data Figs. 12). In contrast, several types of both primary and secondary bile acids were significantly enhanced in the serum and livers of high heat-exposed mice compared with cold- or RT-exposed groups (Extended Data Figs. 13 and 14). In addition, both primary and secondary bile acids were significantly reduced in the serum of high heat-exposed LF-fed or Abx-treated mice compared with high heat-exposed control mice (Extended Data Fig. 15).

Bile acids-TGR5 axis protects from influenza

Thus far, our data indicated that high heat-exposed mice increased host resistance to a broad range of viral infections including influenza A virus and SARS-CoV-2. In addition, high heat-exposed mice may increase gut microbial biotransforming reactions to produce the secondary bile acids. These observations led us to focus on the role of bile acids in resistance to influenza virus infection. We hypothesized that elevated levels of bile acids in the serum of high heat-exposed mice may play an important role in host resistance to viral infection. To address these possibilities, we infected MDCK cells with influenza virus in the presence or absence of various bile acids and found that DCA inhibited the virus protein synthesis (Fig. 3a). Consistent with this observation, influenza virus replication was significantly inhibited by DCA at 24 h p.i. (Fig. 3b). After infection with influenza virus, RT-exposed mice that had been given deoxycholic acid (DCA), cholic acid (CA), ursodeoxycholic acid (UDCA), or taurine-conjugated deoxycholic acid (TDCA) in drinking water for 7 days prior to infection had improved survival relative to control mice (Fig. 3c and Extended Data Fig. 16a-c). This observed protection was associated with improved antiviral resistance to influenza virus infection because the virus titers were significantly suppressed in the lungs of DCA-, CA, or UDCA-treated mice at early time points (Fig. 3d and Extended Data Fig. 16d, e). In CA-treated mice, the levels of TDCA, glycine-conjugated DCA (GDCA) and glycine-conjugated CA (GCA) but not DCA and UDCA in the serum were significantly elevated compared with water-fed control group (Extended Data Fig. 17), suggesting that not only DCA but also taurine- or glycine-conjugated bile acids in serum play an important role in protection against influenza virus infection.

Since bile acids are digestive surfactants that promote lipid absorption, it is possible that they directly disrupt enveloped viruses including influenza virus and SARS-CoV-2 by destroying membrane integrity²⁷. To test this possibility, influenza virus, SARS-CoV-2, or encephalomyocarditis virus (EMCV), a non-enveloped RNA virus, were incubated with various concentrations of DCA at 37 °C for 1 h. Indeed, DCA inactivated the influenza virus and SARS-CoV-2, but not EMCV, at 1.25 mM or higher (Extended Data Fig. 18a-c). However, concentrations of DCA higher than 0.25 mM were toxic to cultured cells (Extended Data Fig. 18d, e).

Several other possible mechanisms could explain how lower concentration of DCA in the serum might increase host resistance to influenza virus infection. First, detergent properties of bile acids might affect viral entry or budding by modulating the integrity of cellular membranes^28,29 (Extended Data Fig. 19a). Second, bile acids activated receptors might regulate virus-induced inflammatory responses^30-32. First, we examined the direct effects of DCA on influenza virus replication. In the presence of 0.125 mM of DCA, the ratio of influenza virus nucleoprotein (NP)-positive cells was significantly enhanced at 6 h p.i. (Extended Data Fig. 19b, c), which indicated that DCA facilitates influenza virus entry into MDCK cells. However, influenza virus replication was significantly inhibited by DCA at 24 and 48 h p.i. (Extended Data Fig. 19d, e). Next, we wished to determine which bile acid receptors could contribute to inhibition of influenza virus replication. To this end, we infected MDCK cells with influenza virus in the presence or absence of GW 4064 (a FXR agonist) or HY-14229 (a TGR5 agonist) and found that 1 μM of HY-14229 but not GW 4064 inhibited influenza virus replication (Fig. 3e, f and Extended Data Fig. 20). In addition, treatment of the virus-infected cells with 1 μM of HY-14229 by 9 h p.i. inhibited influenza virus replication (Extended Data Fig. 21). Further, HY-14229-treated mice significantly reduced the virus replication at 2 d p.i. and had improved survival relative to water-fed group (Fig. 3g, h).

Next, we tested the second hypothesis that bile acids activated receptors might regulate virus-induced inflammatory responses^30-32. High heat-exposed mice impaired secretion of mature IL-1β in the lung wash following influenza virus infection (Extended Data Fig. 22)¹¹. One of the hallmarks of IL-1 signaling is the recruitment of neutrophils^33,34. In addition, previous studies have indicated the detrimental role of neutrophils in influenza virus-induced mortality^3,35. Thus, we first examined the recruitment of neutrophils into the lung of high heat-exposed mice during influenza virus infection. Following influenza virus infection, the levels of pulmonary CXCL1, which is one of the major chemoattractant of neutrophils, in the lung of RT-exposed mice became apparent starting around day 3 p.i. and peaking around day 4 p.i. (Fig. 4a). In contrast, the levels of pulmonary CXCL1 were significantly suppressed in the lung of high-heat exposed mice compared with RT-exposed group at 4 d p.i. (Fig. 4b). Consistent with this observation, both frequency and number of neutrophils were significantly suppressed in the lung of high-heat exposed mice compared with RT-exposed group at 7 d p.i. (Fig. 4c, d). In addition, the levels of CXCL1 and neutrophil recruitment were significantly elevated in the lung of high heat-exposed LF-fed and Abx-treated mice compared with high heat-exposed control group (Extended Data Fig. 23). We next examined whether bile acids or their receptors inhibit influenza virus-induced CXCL1 production. Treatment of bone marrow-derived macrophages (BMMs) with 125 µM of DCA (LDH release <1%) but not CA significantly inhibited not only IL-1β, an important mediator of CXCL1 production (Extended Data Fig. 24)³⁶, but also CXCL1 production from influenza virus-infected BMMs (Fig. 4e-g). Consistent with this observation, RT-exposed mice that had been given DCA in drinking water suppressed neutrophil recruitment into the lung tissue compared with RT-exposed water-fed group (Fig. 4h). In addition, HY-14229 but not GW 4064 inhibited influenza virus-induced IL-1β and CXCL1 production from BMMs (Fig. 4i, j). Further, depletion of neutrophils or neutrophil extracellular traps digestion in vivo resulted in prolonged survival of RT-exposed mice after influenza virus infection (Fig. 4k, l), consistent with previous reports^3,23,37. Together, these data suggested that elevated levels of secondary bile acids in the serum of high heat-exposed mice may increase host resistance to viral infection by suppressing influenza virus replication and detrimental inflammatory responses.

Role of bile acids in COVID-19

We next examined inhibitory effect of bile acids against SARS-CoV-2 infection. To this end, we infected VeroE6/TMPRSS2 cells with SARS-CoV-2 in the presence or absence of various bile acids and found that DCA and UDCA inhibited the virus protein synthesis (Fig. 5a). Consistent with this observation, SARS-CoV-2 replication was significantly inhibited by DCA at 48 h p.i. (Fig. 5b). In addition, DCA-treated hamsters had improved survival relative to water-fed group following SARS-CoV-2 B.1.1.7 (alpha) variant infection without affecting the virus titer at 3 d p.i. (Fig. 5c and Extended Data Fig. 25a, b). Although CA did not inhibit SARS-CoV-2 replication in vitro (Fig. 5a), CA-treated hamsters had improved survival relative to water-fed group after infection with SARS-CoV-2 B.1.1.7 (alpha) variant (Fig. 5d and Extended Data Fig. 25c). Cold-exposed hamsters, which exacerbated SARS-CoV-2-induced mortality (Extended Data Fig. 4), exhibited significant decrease in basal body temperature compared with RT-exposed group (Extended Data Fig. 26a). In addition, cold-exposed naïve hamsters significantly reduced the levels of glycine-conjugated chenodeoxycholic acid (GCDCA), GDCA, and GCA in the serum compared with RT-exposed group (Extended Data Fig. 26b-d). Together, these data suggested that not only DCA but also taurine- or glycine-conjugated bile acids may play an important role in protection against SARS-CoV-2 infection. Interestingly, we found that 100 μM of GW 4064 efficiently blocked SARS-CoV-2 replication in vitro (Fig. 5e and Extended Data Fig. 27). In addition, GW 4064 but not HY 14229 treatment significantly improved survival of hamsters following SARS-CoV-2 B.1.1.7 (alpha) variant infection without affecting the virus titer at early time points (Fig. 5f, g and Extended Data Fig. 28).

Finally, we examined the relationship between the levels of bile acids in the plasma of COVID-19 patients and severity of the disease. To this end, patients who had been admitted to Juntendo University Hospital were stratified into minor or moderate I/II disease groups on the basis of optimal oxygen saturation, clinical symptoms and chest radiographic findings. The levels of serum amyloid A and fibrinogen, important biomarkers of COVID-19 disease severity, were significantly elevated in the plasma of patients who developed moderate I/II disease compared with minor illness group (Fig. 5h and Extended Data Fig. 29). Importantly, the levels of GCA, taurine-conjugated CA (TCA), taurine-conjugated chenodeoxycholic acid (TCDCA), and GCDCA were significantly reduced in the plasma of COVID-19 patients who developed moderate I/II disease compared with minor illness group (Fig. 5i and Extended Data Fig. 30). In addition, the levels of fibrinogen in plasma were significantly and negatively correlated with the levels of the GCA, TCA, TCDCA, and GCDCA in plasma (Extended Data Fig. 31). Treatment of VeroE6/TMPRSS2 cells with 2 mM of GCA (LDH release <1%) significantly inhibited the viral protein synthesis and replication at 48 h p.i. (Extended Data Figs. 32). In addition, GCA-treated hamsters had improved survival relative to water-fed group following SARS-CoV-2 B.1.1.7 (alpha) variant infection (Fig. 5j, k). Taken together, our data show that body temperature-dependent activation of bile acid metabolism by gut microbiota increases host resistance to influenza virus and SARS-CoV-2 infection by suppressing virus replication and detrimental inflammatory responses through bile acid receptors.

Our findings here have identified an unappreciated link between core body temperature, gut microbial activity, and host resistance to viral infection. Specifically, high heat-exposed mice increased their basal body temperature over 38 °C, which may stimulate gut microbial activity³⁸. Indeed, both primary and secondary bile acids were significantly enhanced in the serum and livers of high heat-exposed mice. Increased mortality in partially hepatectomized mice following influenza virus infection may be explained by the fact that primary bile acids are synthesized from cholesterol exclusively in the liver and further metabolized by the gut microbiota into secondary bile acids³⁹. Because secondary bile acids are produced by microbial biotransforming reactions in the gut⁴⁰, high heat-exposed LF-fed and Abx-treated mice significantly reduced the secondary bile acids in the serum and succumbed to influenza virus infection. We have also shown that DCA and HY-14229 (a TGR5 agonist) inhibit influenza virus replication and inflammatory responses both in vitro and in vivo.

It has increasingly evident that gut microbiota promotes type I IFNs-dependent innate antiviral immunity and confers host resistance to influenza virus infection. We and others have previously shown that Abx-treated mice are more susceptible to influenza virus infection^9,11,41,42. Abt et al. have shown that alveolar macrophages isolated from Abx-treated mice decreased innate antiviral genes expression in the lung at 3 d post influenza virus infection. Thus, intranasal administration of Abx-treated mice with poly(I:C) at -1 and 3 d post influenza virus infection improved survival relative to control group⁴¹. In addition, bone marrow chimera experiments revealed that gut microbiota-driven IFN priming in the lungs acts on stromal cells to establish an antiviral state during influenza virus infection⁴². Further, Steed et al. have identified desaminotyrosine as a gut microbiota-derived metabolite that is able to increase interferon-stimulated genes expression in the lungs and host resistance to influenza virus infection⁹. Recent studies indicated that bile acids also increase type I IFN-dependent innate antiviral immunity^43,44. Hu et al. have indicated that both HSV-1 (DNA virus) and Sendai virus (RNA virus) induces accumulation of intracellular bile acids. The accumulated intracellular bile acids activate SRC kinase through the TGR5-GRK-β-arrestin axis to enable tyrosine phosphorylation of multiple innate antiviral signaling components. Therefore, TGR5 knockout mice were more susceptible to HSV-1 and EMCV infections⁴³. Winkler et al. have shown that dysbiosis of gut microbiota increases virus burden in the serum and spleen of Chikungunya virus (CHIKV)-infected mice. However, reconstitution with a single bacterial species, Clostridium scindens, or its derived DCA can restore TLR7 and MyD88-dependent type I IFN responses to restrict systemic CHIKV infection⁴⁴. Although the roles of gut microbiota or its derived secondary bile acids in type I IFN-dependent innate antiviral immune responses are well-established, role of core body temperature in gut microbial activity and host resistance to influenza virus infection remains unknown. We found that high body temperature over 38 °C significantly increased gut microbiota-derived bile acids in high-heat exposed mice. In addition, our study identifies several possible mechanisms by which a gut microbiota-derived secondary bile acid DCA can increase host resistance to influenza virus infection. In addition, we showed that HY-14229 (a TGR5 agonist)-treated RT-exposed mice improved survival following influenza virus infection. Several possible mechanisms could explain how DCA or TGR5 signaling increased host resistance to influenza virus infection. First, detergent properties of DCA at high concentrations (1.25 mM or higher) directly disrupted influenza virus by destroying envelope membrane (Extended Data Fig. 19a, b)²⁷. Second, a low concentration of DCA (125 μM) inhibited influenza virus replication by modulating the integrity of cellular membranes^28,29 (Extended Data Fig. 20a). Third, TGR5 may suppress influenza virus replication (Fig. 3e-g) probably by inhibiting NF-κB activity or adhesion molecule expression⁴⁵. Fourth, DCA and TGR5 signaling suppressed neutrophil recruitment into the lung tissue by inhibiting influenza virus-induced IL-1β and CXCL1 production from macrophages (Fig. 4e, f, h-j). These data are consistent with a published report showing that bile acids including DCA inhibit the NLRP3 inflammasome-mediated IL-1β secretion from BMMs by stimulating TGR5-cAMP-protein kinase A axis-dependent NLRP3 phosphorylation and ubiquitination³¹. Since excess neutrophil recruitment into the lung exacerbated influenza virus-induced mortality^3,35, our data suggests that high body temperature-triggered bile acids production by the gut microbiota may increase host resistance to viral infection by suppressing influenza virus replication and detrimental inflammatory responses.

Our novel findings obtained from influenza virus infection applies to SARS-CoV-2 infection. Specifically, high heat-exposed K18-hACE2 mice were highly resistant to SARS-CoV-2 infection compared with RT-exposed group (Extended Data Fig. 5). In addition, CA- or DCA-treated hamsters had improved survival relative to water-fed group following SARS-CoV-2 B.1.1.7 (alpha) variant infection (Fig. 5c, d). While GW 4064 (a FXR agonist) inhibited SARS-CoV-2 replication in vitro (Fig. 5e and Extended Data Fig. 27), the GW 4064 treatment significantly improved survival of hamsters following SARS-CoV-2 B.1.1.7 (alpha) variant infection without affecting the virus titer at early time points (Fig. 5f, g and Extended Data Fig. 28). Importantly, we found that the levels of GCA, TCA, TCDCA, and GCDCA were significantly reduced in the plasma of COVID-19 patients who developed moderate I/II disease compared with minor illness group (Fig. 5i and Extended Data Fig. 30). In addition, GCA-treated hamsters had improved survival relative to water-fed group following SARS-CoV-2 B.1.1.7 (alpha) variant infection (Fig. 5j, k). These observations suggest that secondary bile acids and their receptor signaling may increase host resistance to SARS-CoV-2 infection. Recent studies suggested that gut microbiome dysbiosis during COVID-19 is associated with detrimental host inflammatory responses and severity of the disease^46,47. Given that the severity of COVID-19 disease is associated with an influx of innate immune cells and inflammatory cytokines^48-50, inhibition of detrimental inflammatory responses by a FXR ligand or probiotic therapy may increase host resistance to SARS-CoV-2 infection.

In summary, our findings substantially expand our understanding of how core body temperature increases host resistance to influenza virus and SARS-CoV-2 infection. Our detailed mechanistic analysis demonstrates that high body temperature-dependent activation of gut microbiota metabolism increases the levels of bile acids in the serum and intestine, and suppresses virus replication and detrimental inflammatory responses following influenza virus and SARS-CoV-2 infection. Our finding that reduction of certain bile acids in the plasma of patients with moderate I/II COVID-19 may provide insight into the variability in clinical disease manifestation in humans and enable approaches for mitigating COVID-19 outcomes.

Methods and any associated references are available in the online version of the paper.

ACKNOWLEDGMENTS

We thank Akiko Iwasaki (Yale University) for critical reading of the manuscript, Yoshihiro Kawaoka (University of Wisconsin and University of Tokyo) for providing SARS-CoV-2/UT-NCGM02/Human/2020/Tokyo, Masayuki Saijo (National Institute of Infectious Diseases) for providing the SARS-CoV-2 QK002 variant, and Chisato Kameda for technical support. Flow cytometric analysis was performed in the IMSUT FACS Core laboratory. This work was supported in part by research grants from JSPS KAKENHI (22H03541 to S.F.), AMED-CREST (JP22gm1010009 to S.F.), JST ERATO (JPMJER1902 to S.F.), the Food Science Institute Foundation (to S.F.), the MSD Life Science Foundation (to T.I.), Public Interest Incorporated Foundation (to T.I.), the Takeda Science Foundation (to T.I.), and the Suzuken Memorial Foundation (to T.I.). M. Moriyama is the Research Fellow of the Japan Society for the Promotion of Science.

AUTHOR CONTRIBUTIONS

S.F. and T.I. conceived the study and designed the experiments; M.N., M.M. Y.N., N.A., M.Ni. and T.I. performed animal experiments; T.I., A.H. and S.F. conducted metabolome analysis; H.M. and T.N. collected clinical samples; C.I., H.W., T.N., T.Y. analyzed data; I.K., D.I. and A.N. provided reagents and advice; and S.F. and T.I. wrote the paper.

COMPETING INTERESTS

T.N., T.Y., D.I. and S.F. were founders and board members of Metagen Therapeutics, Inc., a company involved in the microbiome-based medical and drug discovery. The other authors declare no competing interests.

DATA AVAILABILITY

The authors declare that the data supporting the findings of this study are available within the article and its Supplementary Information files, or are available on request.

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Ethics statement

All protocols involving specimens from human subjects recruited at Juntendo University were reviewed and approved by the Institutional Review of Juntendo University (H20-0222). All animal experiments were performed in accordance with University of Tokyo’s Regulations for Animal Care and Use, which were approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo (PA15-92, PA19-87, PA21-37).

Patients

This is a case-control monocentric study that included 46 patients with COVID-19, hospitalized in the Juntendo University Hospital. All patients were recruited from October 2020 to March 2021. Reverse transcription-polymerase chain reaction (RT-PCR) tests using nasal or pharyngeal swabs were performed for diagnosis of COVID-19. This study was approved by the Ethics Committee of Juntendo University (No. H20-0222) and written informed consent was obtained from all participants. Electronic medical records of the patients’ clinical characteristics on admission were collected. Data included self-reported symptoms before PCR testing, the patient’s medical history, comorbidities, medical treatment received, blood examination result on admission, and chest x-ray and CT results. Data on COVID-19 management were collected as well. Self-reported symptoms on the day of the PCR test were collected via a questionnaire. All imaging and blood test results were collected on admission.

Animals

Age- and sex-matched C57BL/6J or 4-weeks-old female Syrian hamsters obtained from Japan SLC, Inc. were used as WT controls. B6.Cg-Tg(K18-ACE2)2Prlmn/J (K18-hACE2) mice were purchased from The Jackson Laboratory and subsequently bred at The University of Tokyo.

Antibiotic treatment

Mice were treated for 2 weeks with ampicillin (1 g/L; Nacalai Tesque), vancomycin (500 mg/L; Duchefa Biochemie), neomycin sulfate (1 g/L; Nacalai Tesque), metronidazole (1 g/L; Nacalai Tesque), gentamicin (10 mg/L; Nacalai Tesque), 1% penicillin-streptomycin (P/S) (Nacalai Tesque, 09367-34), and 1% amphotericin B (Nacalai Tesque, 02892-54) in drinking water as previously described^11,51. Antibiotic-containing water was changed twice a week.

Cell culture

Madin-Darby canine kidney (MDCK) cells were maintained in minimal essential medium (MEM) (Nacalai Tesque, 21443-15) supplemented with 10% fetal bovine serum (FBS) and 1% P/S. VeroE6 cells stably expressing transmembrane protease serine 2 (VeroE6/TMPRSS2; JCRB Cell Bank 1819) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (low-glucose) (Nacalai Tesque, 08456-65) supplemented with 10% FBS, 1% P/S, and G418 (1 mg ml^-1; Nacalai Tesque, 16512-94). L929 cells were maintained in DMEM (high-glucose) (Nacalai Tesque, 08458-45) supplemented with 10% FBS and 1% P/S. To prepare BMMs, bone marrows from the tibia and femur were obtained by flushing with DMEM. Bone marrow cells were cultured with DMEM supplemented with 10% FBS, L-glutamine, 1% P/S, and 30% L929 supernatant containing the macrophage colony-stimulating factor at 37 °C for 5 days⁵².

Virus infection in vivo

A mouse-adapted influenza A virus strain A/Puerto Rico/8/1934 (PR8) and a human isolate of the 2009 pandemic influenza A virus strain A/Narita/1/2009 (pdm09) were grown in allantoic cavities of 10-d-old fertile chicken egg for 2 d at 35 °C. Viral titers were quantified by a standard plaque assay using MDCK cells and viral stock was stored at -80 °C. For influenza virus infection, mice were infected by intranasal application of 30 μL of virus suspension (500–2,000 pfu of influenza virus in PBS) under isoflurane anaesthesia. An original SARS-CoV-2 strain bearing aspartic acid at position 614 of spike (S) protein (S-614D) and SARS-CoV-2 B.1.1.7 (alpha) variant were grown in VeroE6/TMPRSS2 cells for 2 d at 37 °C. Viral titers were quantified by a standard plaque assay using VeroE6/TMPRSS2 cells and viral stock was stored at -80 °C. For SARS-CoV-2 infection, K18-hACE2 mice were infected by intranasal application of 50 μL of virus suspension (5×10⁴ pfu of SARS-CoV-2 in PBS) under anaesthesia. Syrian hamsters were infected by intranasal application of 150 μL of virus suspension (1.5×10⁶ pfu of SARS-CoV-2 in PBS) under anaesthesia. ZIKV (ATCC VR-84) was grown in Vero cells for 2 d at 37 °C. Viral titers were quantified by a standard plaque assay using Vero cells and viral stock was stored at -80 °C. For ZIKV infection, mice were injected intravenously with 1.5×10⁷ pfu of ZIKV in PBS under anaesthesia. SFTSV (a gift from S. Morikawa, National Institute of Infectious Diseases, Tokyo, Japan) was grown in Vero cells for 3 d at 37 °C. Viral titers were determined by 50% tissue culture infectious dose (TCID₅₀) assay according to published procedures⁵³ and viral stock was stored at -80 °C. For SFTSV infection, mice were injected intravenously with 5×10⁶ TCID₅₀ of SFTSV in PBS under anaesthesia. EMCV was grown in L929 cells for 15 h at 37 °C. Viral titers were quantified by a standard plaque assay using L929 cells and viral stock was stored at -80 °C. For EMCV infection, mice were injected intraperitoneally with 100 pfu of EMCV in PBS under anaesthesia.

Core body temperature was measured by using a rodent thermometer (Natsume Seisakusho, KN-91-AD1687) before and after infection. Baseline body weights were measured before infection. Mice and hamsters were deemed to have reached endpoint at 70% of starting weight or after reaching body temperature of 25 °C or lower. All experiments with SARS-CoV-2 were performed in enhanced biosafety level 3 (BLS-3) containment laboratories at the University of Tokyo, in accordance with the institutional biosafety operating procedures.

Virus infection in vitro

MDCK cells in 6-well plates were infected with influenza virus PR8 at a multiplicity of infection (MOI) of 0.01 in the presence or absence of bile acids, GW 4064 (a FXR agonist), or HY-14229 (a TGR5 agonist) for 1 h at 37 °C, and cultured with Opti-MEM (Invitrogen, 31985-070) containing acetyltrypsin (10 mg/mL) in the presence or absence of bile acids, GW 4064, or HY-14229 for additional 23 h. VeroE6/TMPRSS2 cells in 6-well plates were infected with SARS-CoV-2 at an MOI of 0.01 in the presence or absence of bile acids, GW 4064, or HY-14229 for 1 h at 37 °C, and cultured with DMEM (low-glucose) supplemented with 5% FBS, 1% P/S, and 1% HEPES buffer (Nacalai Tesque, 17557-94) in the presence or absence of bile acids, GW 4064, or HY-14229 for additional 23 h.

Western blot analysis

The virus-infected MDCK or VeroE6/TMPRSS2 cells in 6-well plates were washed with PBS and lysed in 500 μl of 1× TNT buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol). Lysates were centrifuged at 20,630 ×g for 10 min at 4 °C. The supernatant was mixed with 4× lithium dodecyl sulfate (LDS) sample buffer (Invitrogen, NP0007) and 10× sample reducing agent (Invitrogen, NP0009). Samples were boiled for 5 min and fractionated by NuPAGE 10% Bis-Tris Protein Gels (Invitrogen, NP0316BOX) and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, 170-4156). The membranes were incubated with mouse anti-influenza A virus NS1 (Santa Cruz, sc-130568), mouse anti-influenza A virus M2 (Santa Cruz, sc-32238), mouse anti-α tubulin (Santa Cruz, sc-32293), or rabbit anti-SARS-CoV-2 nucleocapsid (Cell Signaling, 33336) antibody, followed by incubation with horseradish peroxidase-conjugated anti-mouse IgG (Jackson Immuno Research Laboratories, 115-035-003) or anti-rabbit IgG (Invitrogen, G-21234). The PVDF membranes were then treated with Chemi-Lumi One Super (Nacalai Tesque, 02230-30) to elicit chemiluminescent signals, which were detected and visualized using an LAS-4000 Mini apparatus (GE Healthcare).

Quantitative PCR

Total RNA was extracted from cells using TRIzol reagent (Invitrogen, 15596018) and reverse transcribed into cDNA using SuperScript III reverse transcriptase (Invitrogen, 18080085) with an oligo (dT) primer. TB Green Premix Ex Taq II (TaKaRa, RR820A) and a LightCycler instrument (Roche Diagnostics) were used for quantitative PCR with the following primers: influenza virus NP forward, 5’-agaacatctgacatgaggac-3’, and reverse, 5’-gtcaaaggaaggcacgatc-3’.

ELISA

Cell-free supernatants or lung washes were analyzed for the presence of IFN-α (Hycult Biotech, HM1001; PBL Assay Science, 32100-1), IFN-γ (eBioscience, 14-7311-85 and 13-7312-85,), IL-1β (eBioscience, 14-7012-85 and 13-7112-85), and IL-6 (eBioscience, 14-7061-85 and 13-7062-85) using an enzyme-linked immunosorbent assay (ELISA) utilizing paired antibodies. IFN-β (PBL Assay Science, 42400-1), IFN-λ (PBL Assay Science, 62830-1), or CXCL1 (Proteintech, KE10019) ELISA was performed according to the manufacturer’s instructions.

LDH releases assay

LDH release assays (Promega, G1780) were performed according to the manufacturer’s instructions. LDH release data were used to account for cell death. The data are expressed as percentage of maximum LDH release.

Flow cytometry

The single-cell suspensions of lung samples were prepared as previously described⁵⁴. For neutrophil staining, cells were incubated with APC-labeled anti-Ly6G (Invitrogen, 17-9668-82) and eFluor 450-labeled anti-Ly6C (Invitrogen, 48-5932-82). For the detection of influenza virus-infected cells, MDCK cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences, 554714), and intracellulary stained with FITC-labeled mouse anti-influenza virus NP (abcam, ab20921) antibody. Flow cytometric analysis was performed with a FACSVerse flow cytometer (BD Biosciences). The final analysis and graphical output were performed using FlowJo software (Tree Star, Inc.).

Metabolite extraction and CE-TOFMS measurements

Metabolites in the cecal and serum samples were analyzed as previously described^55,56. Briefly, cecal and serum metabolites were extracted by vigorous shaking with methanol containing methionine sulfone and D-camphol-10-sulfonic acid as the internal standards. Then the mixture was cleaned with chloroform and water extraction. The suspension was centrifuged at 4,600 × g for 15 min at 4˚C, and the resulting supernatant was transferred to a 5-kDa-cutoff filter column (Ultrafree MC-PLHCC 250/pk for Metabolome Analysis, Human Metabolome Technologies, Tsuruoka, Yamagata, Japan). The flow-through was dried under vacuum, and the residue then was dissolved in Milli-Q water containing reference compounds (200 μM each of 3-aminopyrrolidine and trimesate). The levels of extracted metabolites were measured in both positive and negative modes by CE-TOFMS as previously described⁵⁷. All CE-TOFMS experiments were performed using an Agilent capillary electrophoresis system and Agilent G3250AA LC/MSD TOF system (Agilent Technologies, Santa Clara, California, U.S.A).

Principal component analysis of CE-TOFMS data was performed using SIMCA 13.0 (Umetrics, Umea, Sweden). For the analysis, concentrations below the detection limit were substituted with zero, and metabolites whose levels were below the detection limit in all the samples were excluded.

Quantification of serum and liver bile acids via LC-MS/MS

Murine serum and liver metabolites were extracted from 50 μL serum or 50 μg liver suspended in 250 µL of 50% methanol in Milli-Q water supplemented with 20 μM CSA as an internal standard. The samples were homogenized using a multi-sample homogenizer (Shake Master Neo, Bio Medical Science) and centrifuged at 20,400 × g for 10 min at 10 °C, and supernatants were harvested. Liquid chromatography was performed using an Agilent 1290 UPLC system (Agilent Technologies) with gradient elution from an ACQUITY UPLC HSS T3 column (1.8 µm, 50 mm×2.1 mm ID; Waters) maintained at 45 °C with a mobile phase flow rate of 0.3 ml/min ⁵⁸. Gradient elution mobile phases comprised A (water, 50 mM ammonium formate) and B (methanol, 50 mM ammonium formate). The gradient was initiated at 10% B, increasing linearly to 50% at 0.1 min, held at 50% B at 2 min, increasing linearly again to 80% at 9.5 min, and then increasing linearly to 100% at 10.5 min; this was maintained until 12 min, with subsequent re equilibration at 10% B for a further 5 min. The sample temperature was maintained at 4 °C in the autosampler prior to analysis. The sample injection volume was 1 µL. Mass spectrometry (MS) analysis was performed using an Agilent 6490 triple quadrupole mass spectrometer MS (Agilent Technologies) equipped with an Agilent Jet Stream ESI probe in negative-ion mode. A capillary voltage of -3,500 V, a source temperature of 200 °C, gas flow of 14 L/min, nebulizer of 50 psi, sheath gas temperature of 250 °C, and a sheath gas flow of 11 L/min were used. Optimized m/z values of precursor and product ions, dwell times, collision voltages, and retention times selected for each compound are indicated previously⁵⁹. Concentrations of individual BAs were determined from the peak area in the chromatogram detected with MRM relative to the internal standard, CSA.

Quantification of cecal organic acids via GC-MS

Organic acid concentrations of caecal contents were determined by gas chromatography–mass spectrometry (GC–MS)⁶⁰. In brief, 10 mg cecal contents were disrupted using 3 mm zirconia/silica beads (BioSpec Products) and homogenized with extraction solution containing 100 μl of internal standard (100 μM crotonic acid), 50 μl HCl and 200 μl ether. After vigorous shaking using Shakemaster neo (Bio Medical Science) at 1,500 r.p.m. for 10 min, homogenates were centrifuged at 1,000g for 10 min and then the top ether layer was collected and transferred into new glass vials. Aliquots (80 μl) of the ether extracts were mixed with 16 μl N-tert-butyldimethylsilyl-Nmethyltrifluoroacetamide (MTBSTFA). The vials were sealed tightly, heated at 80 °C for 20 min in a water bath, and then left at room temperature for 48 h for derivatization. The derivatized samples were run through a 6890N Network GC System (Agilent Technologies) equipped with HP-5MS column (0.25 mm x 30 m x 0.25 μm) and 5973 Network Mass Selective Detector (Agilent Technologies). Pure helium (99.9999%) was used a carrier gas and delivered at a flow rate of 1.2 ml per min. The head pressure was set at 97 kPa with split 20:1. The inlet and transfer line temperatures were 250 and 260 °C, respectively. The following temperature program was used: 60 °C (3 min), 60–120 °C (5 °C per min), 120–300 °C (20 °C per min). One microliter of each sample was injected with a run time of 30 min. Organic acid concentrations were quantified by comparing their peak areas with the standards.

Statistical analysis

Statistical significance was tested using nonparametric one-way analysis of variance (ANOVA), using PRISM software (version 5; GraphPad software). P values of <0.05 were considered statistically significant.

Yes there is potential Competing Interest. T.N., T.Y., D.I. and S.F. were founders and board members of Metagen Therapeutics, Inc., a company involved in the microbiome-based medical and drug discovery. The other authors declare no competing interests.

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High body temperature increases gut microbiota-dependent host resistance to influenza A virus and SARS-CoV-2 infection

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