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Organisms adapt to day-night cycles through highly specialized circadian machinery, whose molecular components anticipate and drive changes in organism behavior and metabolism. Although many effectors of the immune system are known to follow daily oscillations, the role of the circadian clock in the immune response to acute infections is not unders...

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... model to determine how the circadian clock regulates the host response during acute Salmonella infection. Differential Day – Night Response to Salmonella Infection. To determine whether the circadian clock regulates the host response to infection with S. Typhimurium, we infected wild-type (WT) mice by oral gavage either at 10:00 AM (day, early rest phase; zeitgeber time 4, ZT4) or at 10:00 PM (night, early active phase; ZT16). At 48, 60, 72, and 78 h postinfection (p.i.), mice were killed, and tissue samples collected for bacteriology, histopathology, and gene expression analyses (Fig. 1). We observed that S. Typhimurium colonization signi fi cantly changed with time of infection, espe- cially at later time points. Notably, S. Typhimurium numbers were signi fi cantly increased in the colon content of mice infected at 10:00 AM in comparison with 10:00 PM at both 72 and 78 h p.i. (Fig. 1 A ); colonization of Peyer ’ s patches and spleen was also signi fi cantly higher in mice infected during the day (Fig. S1 A ). Next, we determined whether the degree of the host response to infection changed with the time of inoculation. Histopathology showed that ceca from mice infected during the day were on average more in fl amed than those from mice infected during the night, at 48, 72, and 78 h p.i. (Fig. 1 B and C ; Figs. S1 B and S2). In contrast, at 60 h p.i., a mild increase in in fl ammation was observed in mice infected at night, suggesting that the time of infection was not the only variable in fl uencing the in fl ammatory response. Reduced signs of cecal in fl ammation were characterized by low-grade submucosal edema and neutrophil in fl ux (Fig. 1 B and C and Figs. S1 B and S2). Major differences were also found in the levels of cryptitis and surface erosions (Fig. S1 B ), which constitute other classical signs of in fl ammation (10 – 12). To ascertain whether these differences were accompanied by changes in gene expression, we analyzed expression of proin fl ammatory cytokine Tnf α , neutrophil chemoattractant chemokine (C-X-C motif) ligand 1 ( Cxcl-1 ), and antimicrobial peptide lipocalin-2 ( Lcn-2 ) in the cecum of infected and uninfected mice and found signi fi cant differences during the course of infection (Fig. 1 D and Table S1). Consistent with our previous fi ndings (11 – 13), mice infected during the day showed increased expression of these genes compared with uninfected controls, and the expression was dependent both on time of infection and time of death (Fig. 1 D ). Genes in mice infected in the morning (ZT4) or at night (ZT16) showed maximal expression when killed during the day (48 and 72 h for mice infected at ZT4; 60 h for mice infected at ZT16). This fi nding was particularly notable for Tnf α because its expression was increased at both 48 and 72 h in mice infected at ZT4 (ZT4 death) relative to those infected at ZT16 (ZT16 death), and the opposite trend was observed at 60 h (ZT16 death for mice infected at ZT4; ZT4 for mice infected at ZT16). In line with this observation is the absence of signi fi cant changes in expression in mice killed 78 h after infection, corresponding to ZT10 for mice infected at ZT4 and ZT22 for mice infected at ZT16 (Fig. 1 D ). Additionally, the overall expression levels of the three proin fl ammatory genes analyzed were reduced in mice infected at ZT16 (yellow and gray graphs of Fig. 1 D ), consistently with the differences observed in colonization level and pathology score. To establish whether circadian transcription follows a normal pro fi le during infection we analyzed the expression of the clock gene period 2 ( Per2 ). Per2 oscillation in uninfected mice demonstrated proper synchronization (Fig. 1 D ). Notably, Per2 expression was progressively down-regulated after infection (Fig. 1 D ). These results suggest that a circadian mechanism may regulate components of the innate immune response to acute Salmonella infection, also revealing a profound repression of circadian components during infection. Salmonella -Infected Bone Marrow-Derived Macrophages. Our fi nding that the in fl ammatory response to S. Typhimurium infection is time-of-day – dependent in vivo prompted us to determine whether we could reproduce similar results in vitro in a less complex system. As detailed earlier, macrophages are involved in the fi rst response to S. Typhimurium infection. Moreover, macrophages have an ef fi cient clock machinery, and LPS-activated pathways in these cells are under tight circadian regulation at multiple levels (3, 4). However, bone marrow-derived macrophages (BMDMs) do not synchronously oscillate after 1 wk of differentiation in vitro (Fig. S3 A ), and LPS stimulation at different circadian times leads to similar levels of Il-6 expression in these asynchronous cultures (Fig. S3 B ). We therefore tested whether BMDMs could be synchronized by common methods such as dexamethasone or high-serum treatments (Fig. S3 C and D ). Oscillation of circadian genes Per2 (Fig. S3 C and D ), cryptochrome 1 ( Cry1 ) and brain and muscle ARNT-like protein 1 ( Bmal1 ) (Fig. S3 C ) con fi rmed entrainment of these cells. To better evaluate the contribution of the circadian system to the expression of proin fl ammatory genes, we added LPS (1 μ g/mL) to synchronized macrophages at different times of their circadian cycle and followed the expression pro fi le of Il-6 (Fig. S3 D ). As expected, we obtained different curves of expression depending on the time of LPS administration, with minimal induction for administration at T18 or T30 and major induction at T12 or T24, where T0 is the time when synchronization began. Notably, expression levels appear to be dependent both on the time of treatment and on the time of collection, as suggested by our in vivo data. We next ascertained whether disruption of the circadian clock could affect the function of some central component of the LPS response. Circadian gene expression in mice carrying a mutation of the master circadian regulator Clock [ Clock mutant or clock/ clock mice (19)] is both phase-shifted by 8 h as well as reduced compared with WT littermates (20). Clock mutant mice also fail to rhythmically express a number of immunoregulatory genes in the liver (21). Therefore, we followed the timing of expression of different cytokines after LPS stimulation of BMDMs isolated from either WT or Clock mutant mice (Fig. 2). Remarkably, we observed an overall reduction in the fold induction of the proin fl ammatory genes Il-6 , Il-1 β , Tnf α , Cxcl-1 , Ifn- β , and Che- mokine (C-C motif) ligand 2 ( Ccl2 ) in response to LPS stimulation in BMDMs isolated from Clock mutant mice compared with WT mice (Fig. 2 A ); BMDMs isolated from Clock mutant mice also exhibited reduced secretion of IL-6 and TNF- α after 24 h of LPS stimulation (Fig. 2 B ). A comparable reduction in the levels of IL-6 in the supernatant of BMDMs isolated from Clock mutant mice, compared with WT mice, was obtained after infection with S. Typhimurium (Fig. 2 C ). Because the induction of IL-6 is largely dependent on LPS stimulation of the TLR4 pathway, we also infected BMDMs with an isogenic S. Typhimurium strain carrying a mutation in the lipid A acylation pathway ( msbB mutant), which impairs signaling through TLR4 (11). As predicted, secretion of IL-6 was reduced in BMDMs infected with the msbB mutant compared with infection with WT S. Typhimurium. In contrast, both S. Typhimurium WT and the msbB mutant elicited similar low levels of secretion of IL-6 (Fig. 2 C ) in BMDMs isolated from Clock mutant mice, indicating that TLR4 signaling in response to S. Typhimurium was not induced in the absence of a functional clock. Next we sought to in- vestigate whether the secretion of the proin fl ammatory cytokine IL-1 β was also impaired in Clock mutant mice. IL-1 β is directly induced by TNF- α , causing its expression to peak later than Tnf α , and mature IL-1 β secretion requires both TLR4 and NLR signaling (22). Upon S. Typhimurium infection of BMDMs, LPS activation of TLR4 induces pro – IL-1 β expression, which is followed by NLR-mediated signaling through ice protease-activating factor/NLR family CARD domain-containing protein 4 (Ipaf/ Nlrc4) in response to Salmonella T3SS-1 secretion of fl agellin into the cytosol (22). As expected from these earlier studies, S. Typhimurium strains carrying either a mutation in msbB (unable to signal through TLR4), a mutation in both fl agellin genes ( fl iC fl jB mutant, unable to signal through Ipaf/Nlrc4), or a mutation in T3SS-1 ( invA mutant, unable to signal through Ipaf/Nlrc4) induced secretion of lower levels of IL-1 β compared with S. Typhimurium WT (Fig. 2 D ) in BMDMs from WT mice. In contrast, infection of BMDMs from Clock mutant mice with S. Typhimurium WT resulted in a marked reduction of IL-1 β secretion. Moreover, all S. Typhimurium mutants elicited similar low levels of IL-1 β in Clock mutant BMDMs. Col- lectively, we observed that secretion of proin fl ammatory cytokines in Clock mutant BMDMs was particularly low and comparable to levels elicited by S. Typhimurium mutants designed to evade activation of particular components of the in fl ammatory response. Thus, an intact circadian machinery appears to be required for the induction of proin fl ammatory cytokines in vitro in response to Salmonella infection. Mice. Because the circadian clock is necessary for a robust proin- fl ammatory response in isolated macrophages, we explored its in- volvement in the host response to Salmonella in vivo. We infected Clock mutant mice at 10:00 AM (ZT4) or 10:00 PM (ZT16) (Fig. 3 A ). Differently from WT mice, similar numbers of S. Typhimurium were recovered 72 h p.i. (Fig. 3 A ). Similar results were also obtained when Clock -de fi cient mice ( Clock − / − ) and their WT littermates were infected, thus con fi rming that a functional CLOCK protein is necessary for the observed effect. (Fig. S4 A ). Furthermore, a signi fi cant ...
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... (night, early active phase; ZT16). At 48, 60, 72, and 78 h postinfection (p.i.), mice were killed, and tissue samples collected for bacteriology, histopathology, and gene expression analyses (Fig. 1). We observed that S. Typhimurium colonization signi fi cantly changed with time of infection, espe- cially at later time points. Notably, S. Typhimurium numbers were signi fi cantly increased in the colon content of mice infected at 10:00 AM in comparison with 10:00 PM at both 72 and 78 h p.i. (Fig. 1 A ); colonization of Peyer ’ s patches and spleen was also signi fi cantly higher in mice infected during the day (Fig. S1 A ). Next, we determined whether the degree of the host response to infection changed with the time of inoculation. Histopathology showed that ceca from mice infected during the day were on average more in fl amed than those from mice infected during the night, at 48, 72, and 78 h p.i. (Fig. 1 B and C ; Figs. S1 B and S2). In contrast, at 60 h p.i., a mild increase in in fl ammation was observed in mice infected at night, suggesting that the time of infection was not the only variable in fl uencing the in fl ammatory response. Reduced signs of cecal in fl ammation were characterized by low-grade submucosal edema and neutrophil in fl ux (Fig. 1 B and C and Figs. S1 B and S2). Major differences were also found in the levels of cryptitis and surface erosions (Fig. S1 B ), which constitute other classical signs of in fl ammation (10 – 12). To ascertain whether these differences were accompanied by changes in gene expression, we analyzed expression of proin fl ammatory cytokine Tnf α , neutrophil chemoattractant chemokine (C-X-C motif) ligand 1 ( Cxcl-1 ), and antimicrobial peptide lipocalin-2 ( Lcn-2 ) in the cecum of infected and uninfected mice and found signi fi cant differences during the course of infection (Fig. 1 D and Table S1). Consistent with our previous fi ndings (11 – 13), mice infected during the day showed increased expression of these genes compared with uninfected controls, and the expression was dependent both on time of infection and time of death (Fig. 1 D ). Genes in mice infected in the morning (ZT4) or at night (ZT16) showed maximal expression when killed during the day (48 and 72 h for mice infected at ZT4; 60 h for mice infected at ZT16). This fi nding was particularly notable for Tnf α because its expression was increased at both 48 and 72 h in mice infected at ZT4 (ZT4 death) relative to those infected at ZT16 (ZT16 death), and the opposite trend was observed at 60 h (ZT16 death for mice infected at ZT4; ZT4 for mice infected at ZT16). In line with this observation is the absence of signi fi cant changes in expression in mice killed 78 h after infection, corresponding to ZT10 for mice infected at ZT4 and ZT22 for mice infected at ZT16 (Fig. 1 D ). Additionally, the overall expression levels of the three proin fl ammatory genes analyzed were reduced in mice infected at ZT16 (yellow and gray graphs of Fig. 1 D ), consistently with the differences observed in colonization level and pathology score. To establish whether circadian transcription follows a normal pro fi le during infection we analyzed the expression of the clock gene period 2 ( Per2 ). Per2 oscillation in uninfected mice demonstrated proper synchronization (Fig. 1 D ). Notably, Per2 expression was progressively down-regulated after infection (Fig. 1 D ). These results suggest that a circadian mechanism may regulate components of the innate immune response to acute Salmonella infection, also revealing a profound repression of circadian components during infection. Salmonella -Infected Bone Marrow-Derived Macrophages. Our fi nding that the in fl ammatory response to S. Typhimurium infection is time-of-day – dependent in vivo prompted us to determine whether we could reproduce similar results in vitro in a less complex system. As detailed earlier, macrophages are involved in the fi rst response to S. Typhimurium infection. Moreover, macrophages have an ef fi cient clock machinery, and LPS-activated pathways in these cells are under tight circadian regulation at multiple levels (3, 4). However, bone marrow-derived macrophages (BMDMs) do not synchronously oscillate after 1 wk of differentiation in vitro (Fig. S3 A ), and LPS stimulation at different circadian times leads to similar levels of Il-6 expression in these asynchronous cultures (Fig. S3 B ). We therefore tested whether BMDMs could be synchronized by common methods such as dexamethasone or high-serum treatments (Fig. S3 C and D ). Oscillation of circadian genes Per2 (Fig. S3 C and D ), cryptochrome 1 ( Cry1 ) and brain and muscle ARNT-like protein 1 ( Bmal1 ) (Fig. S3 C ) con fi rmed entrainment of these cells. To better evaluate the contribution of the circadian system to the expression of proin fl ammatory genes, we added LPS (1 μ g/mL) to synchronized macrophages at different times of their circadian cycle and followed the expression pro fi le of Il-6 (Fig. S3 D ). As expected, we obtained different curves of expression depending on the time of LPS administration, with minimal induction for administration at T18 or T30 and major induction at T12 or T24, where T0 is the time when synchronization began. Notably, expression levels appear to be dependent both on the time of treatment and on the time of collection, as suggested by our in vivo data. We next ascertained whether disruption of the circadian clock could affect the function of some central component of the LPS response. Circadian gene expression in mice carrying a mutation of the master circadian regulator Clock [ Clock mutant or clock/ clock mice (19)] is both phase-shifted by 8 h as well as reduced compared with WT littermates (20). Clock mutant mice also fail to rhythmically express a number of immunoregulatory genes in the liver (21). Therefore, we followed the timing of expression of different cytokines after LPS stimulation of BMDMs isolated from either WT or Clock mutant mice (Fig. 2). Remarkably, we observed an overall reduction in the fold induction of the proin fl ammatory genes Il-6 , Il-1 β , Tnf α , Cxcl-1 , Ifn- β , and Che- mokine (C-C motif) ligand 2 ( Ccl2 ) in response to LPS stimulation in BMDMs isolated from Clock mutant mice compared with WT mice (Fig. 2 A ); BMDMs isolated from Clock mutant mice also exhibited reduced secretion of IL-6 and TNF- α after 24 h of LPS stimulation (Fig. 2 B ). A comparable reduction in the levels of IL-6 in the supernatant of BMDMs isolated from Clock mutant mice, compared with WT mice, was obtained after infection with S. Typhimurium (Fig. 2 C ). Because the induction of IL-6 is largely dependent on LPS stimulation of the TLR4 pathway, we also infected BMDMs with an isogenic S. Typhimurium strain carrying a mutation in the lipid A acylation pathway ( msbB mutant), which impairs signaling through TLR4 (11). As predicted, secretion of IL-6 was reduced in BMDMs infected with the msbB mutant compared with infection with WT S. Typhimurium. In contrast, both S. Typhimurium WT and the msbB mutant elicited similar low levels of secretion of IL-6 (Fig. 2 C ) in BMDMs isolated from Clock mutant mice, indicating that TLR4 signaling in response to S. Typhimurium was not induced in the absence of a functional clock. Next we sought to in- vestigate whether the secretion of the proin fl ammatory cytokine IL-1 β was also impaired in Clock mutant mice. IL-1 β is directly induced by TNF- α , causing its expression to peak later than Tnf α , and mature IL-1 β secretion requires both TLR4 and NLR signaling (22). Upon S. Typhimurium infection of BMDMs, LPS activation of TLR4 induces pro – IL-1 β expression, which is followed by NLR-mediated signaling through ice protease-activating factor/NLR family CARD domain-containing protein 4 (Ipaf/ Nlrc4) in response to Salmonella T3SS-1 secretion of fl agellin into the cytosol (22). As expected from these earlier studies, S. Typhimurium strains carrying either a mutation in msbB (unable to signal through TLR4), a mutation in both fl agellin genes ( fl iC fl jB mutant, unable to signal through Ipaf/Nlrc4), or a mutation in T3SS-1 ( invA mutant, unable to signal through Ipaf/Nlrc4) induced secretion of lower levels of IL-1 β compared with S. Typhimurium WT (Fig. 2 D ) in BMDMs from WT mice. In contrast, infection of BMDMs from Clock mutant mice with S. Typhimurium WT resulted in a marked reduction of IL-1 β secretion. Moreover, all S. Typhimurium mutants elicited similar low levels of IL-1 β in Clock mutant BMDMs. Col- lectively, we observed that secretion of proin fl ammatory cytokines in Clock mutant BMDMs was particularly low and comparable to levels elicited by S. Typhimurium mutants designed to evade activation of particular components of the in fl ammatory response. Thus, an intact circadian machinery appears to be required for the induction of proin fl ammatory cytokines in vitro in response to Salmonella infection. Mice. Because the circadian clock is necessary for a robust proin- fl ammatory response in isolated macrophages, we explored its in- volvement in the host response to Salmonella in vivo. We infected Clock mutant mice at 10:00 AM (ZT4) or 10:00 PM (ZT16) (Fig. 3 A ). Differently from WT mice, similar numbers of S. Typhimurium were recovered 72 h p.i. (Fig. 3 A ). Similar results were also obtained when Clock -de fi cient mice ( Clock − / − ) and their WT littermates were infected, thus con fi rming that a functional CLOCK protein is necessary for the observed effect. (Fig. S4 A ). Furthermore, a signi fi cant difference was found in the colonization of Peyer ’ s patches between WT mice infected at day and Clock mutant mice, even if these lymphatic structures were signi fi cantly enlarged in Clock mutant mice during infection (Fig. S4 B ). Moreover, the histopathology revealed higher in fl ammation in Clock mutant mice infected at night compared with mice infected during the day (Fig. 3 B and C ; Fig. S4 C ...
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... infection. Differential Day – Night Response to Salmonella Infection. To determine whether the circadian clock regulates the host response to infection with S. Typhimurium, we infected wild-type (WT) mice by oral gavage either at 10:00 AM (day, early rest phase; zeitgeber time 4, ZT4) or at 10:00 PM (night, early active phase; ZT16). At 48, 60, 72, and 78 h postinfection (p.i.), mice were killed, and tissue samples collected for bacteriology, histopathology, and gene expression analyses (Fig. 1). We observed that S. Typhimurium colonization signi fi cantly changed with time of infection, espe- cially at later time points. Notably, S. Typhimurium numbers were signi fi cantly increased in the colon content of mice infected at 10:00 AM in comparison with 10:00 PM at both 72 and 78 h p.i. (Fig. 1 A ); colonization of Peyer ’ s patches and spleen was also signi fi cantly higher in mice infected during the day (Fig. S1 A ). Next, we determined whether the degree of the host response to infection changed with the time of inoculation. Histopathology showed that ceca from mice infected during the day were on average more in fl amed than those from mice infected during the night, at 48, 72, and 78 h p.i. (Fig. 1 B and C ; Figs. S1 B and S2). In contrast, at 60 h p.i., a mild increase in in fl ammation was observed in mice infected at night, suggesting that the time of infection was not the only variable in fl uencing the in fl ammatory response. Reduced signs of cecal in fl ammation were characterized by low-grade submucosal edema and neutrophil in fl ux (Fig. 1 B and C and Figs. S1 B and S2). Major differences were also found in the levels of cryptitis and surface erosions (Fig. S1 B ), which constitute other classical signs of in fl ammation (10 – 12). To ascertain whether these differences were accompanied by changes in gene expression, we analyzed expression of proin fl ammatory cytokine Tnf α , neutrophil chemoattractant chemokine (C-X-C motif) ligand 1 ( Cxcl-1 ), and antimicrobial peptide lipocalin-2 ( Lcn-2 ) in the cecum of infected and uninfected mice and found signi fi cant differences during the course of infection (Fig. 1 D and Table S1). Consistent with our previous fi ndings (11 – 13), mice infected during the day showed increased expression of these genes compared with uninfected controls, and the expression was dependent both on time of infection and time of death (Fig. 1 D ). Genes in mice infected in the morning (ZT4) or at night (ZT16) showed maximal expression when killed during the day (48 and 72 h for mice infected at ZT4; 60 h for mice infected at ZT16). This fi nding was particularly notable for Tnf α because its expression was increased at both 48 and 72 h in mice infected at ZT4 (ZT4 death) relative to those infected at ZT16 (ZT16 death), and the opposite trend was observed at 60 h (ZT16 death for mice infected at ZT4; ZT4 for mice infected at ZT16). In line with this observation is the absence of signi fi cant changes in expression in mice killed 78 h after infection, corresponding to ZT10 for mice infected at ZT4 and ZT22 for mice infected at ZT16 (Fig. 1 D ). Additionally, the overall expression levels of the three proin fl ammatory genes analyzed were reduced in mice infected at ZT16 (yellow and gray graphs of Fig. 1 D ), consistently with the differences observed in colonization level and pathology score. To establish whether circadian transcription follows a normal pro fi le during infection we analyzed the expression of the clock gene period 2 ( Per2 ). Per2 oscillation in uninfected mice demonstrated proper synchronization (Fig. 1 D ). Notably, Per2 expression was progressively down-regulated after infection (Fig. 1 D ). These results suggest that a circadian mechanism may regulate components of the innate immune response to acute Salmonella infection, also revealing a profound repression of circadian components during infection. Salmonella -Infected Bone Marrow-Derived Macrophages. Our fi nding that the in fl ammatory response to S. Typhimurium infection is time-of-day – dependent in vivo prompted us to determine whether we could reproduce similar results in vitro in a less complex system. As detailed earlier, macrophages are involved in the fi rst response to S. Typhimurium infection. Moreover, macrophages have an ef fi cient clock machinery, and LPS-activated pathways in these cells are under tight circadian regulation at multiple levels (3, 4). However, bone marrow-derived macrophages (BMDMs) do not synchronously oscillate after 1 wk of differentiation in vitro (Fig. S3 A ), and LPS stimulation at different circadian times leads to similar levels of Il-6 expression in these asynchronous cultures (Fig. S3 B ). We therefore tested whether BMDMs could be synchronized by common methods such as dexamethasone or high-serum treatments (Fig. S3 C and D ). Oscillation of circadian genes Per2 (Fig. S3 C and D ), cryptochrome 1 ( Cry1 ) and brain and muscle ARNT-like protein 1 ( Bmal1 ) (Fig. S3 C ) con fi rmed entrainment of these cells. To better evaluate the contribution of the circadian system to the expression of proin fl ammatory genes, we added LPS (1 μ g/mL) to synchronized macrophages at different times of their circadian cycle and followed the expression pro fi le of Il-6 (Fig. S3 D ). As expected, we obtained different curves of expression depending on the time of LPS administration, with minimal induction for administration at T18 or T30 and major induction at T12 or T24, where T0 is the time when synchronization began. Notably, expression levels appear to be dependent both on the time of treatment and on the time of collection, as suggested by our in vivo data. We next ascertained whether disruption of the circadian clock could affect the function of some central component of the LPS response. Circadian gene expression in mice carrying a mutation of the master circadian regulator Clock [ Clock mutant or clock/ clock mice (19)] is both phase-shifted by 8 h as well as reduced compared with WT littermates (20). Clock mutant mice also fail to rhythmically express a number of immunoregulatory genes in the liver (21). Therefore, we followed the timing of expression of different cytokines after LPS stimulation of BMDMs isolated from either WT or Clock mutant mice (Fig. 2). Remarkably, we observed an overall reduction in the fold induction of the proin fl ammatory genes Il-6 , Il-1 β , Tnf α , Cxcl-1 , Ifn- β , and Che- mokine (C-C motif) ligand 2 ( Ccl2 ) in response to LPS stimulation in BMDMs isolated from Clock mutant mice compared with WT mice (Fig. 2 A ); BMDMs isolated from Clock mutant mice also exhibited reduced secretion of IL-6 and TNF- α after 24 h of LPS stimulation (Fig. 2 B ). A comparable reduction in the levels of IL-6 in the supernatant of BMDMs isolated from Clock mutant mice, compared with WT mice, was obtained after infection with S. Typhimurium (Fig. 2 C ). Because the induction of IL-6 is largely dependent on LPS stimulation of the TLR4 pathway, we also infected BMDMs with an isogenic S. Typhimurium strain carrying a mutation in the lipid A acylation pathway ( msbB mutant), which impairs signaling through TLR4 (11). As predicted, secretion of IL-6 was reduced in BMDMs infected with the msbB mutant compared with infection with WT S. Typhimurium. In contrast, both S. Typhimurium WT and the msbB mutant elicited similar low levels of secretion of IL-6 (Fig. 2 C ) in BMDMs isolated from Clock mutant mice, indicating that TLR4 signaling in response to S. Typhimurium was not induced in the absence of a functional clock. Next we sought to in- vestigate whether the secretion of the proin fl ammatory cytokine IL-1 β was also impaired in Clock mutant mice. IL-1 β is directly induced by TNF- α , causing its expression to peak later than Tnf α , and mature IL-1 β secretion requires both TLR4 and NLR signaling (22). Upon S. Typhimurium infection of BMDMs, LPS activation of TLR4 induces pro – IL-1 β expression, which is followed by NLR-mediated signaling through ice protease-activating factor/NLR family CARD domain-containing protein 4 (Ipaf/ Nlrc4) in response to Salmonella T3SS-1 secretion of fl agellin into the cytosol (22). As expected from these earlier studies, S. Typhimurium strains carrying either a mutation in msbB (unable to signal through TLR4), a mutation in both fl agellin genes ( fl iC fl jB mutant, unable to signal through Ipaf/Nlrc4), or a mutation in T3SS-1 ( invA mutant, unable to signal through Ipaf/Nlrc4) induced secretion of lower levels of IL-1 β compared with S. Typhimurium WT (Fig. 2 D ) in BMDMs from WT mice. In contrast, infection of BMDMs from Clock mutant mice with S. Typhimurium WT resulted in a marked reduction of IL-1 β secretion. Moreover, all S. Typhimurium mutants elicited similar low levels of IL-1 β in Clock mutant BMDMs. Col- lectively, we observed that secretion of proin fl ammatory cytokines in Clock mutant BMDMs was particularly low and comparable to levels elicited by S. Typhimurium mutants designed to evade activation of particular components of the in fl ammatory response. Thus, an intact circadian machinery appears to be required for the induction of proin fl ammatory cytokines in vitro in response to Salmonella infection. Mice. Because the circadian clock is necessary for a robust proin- fl ammatory response in isolated macrophages, we explored its in- volvement in the host response to Salmonella in vivo. We infected Clock mutant mice at 10:00 AM (ZT4) or 10:00 PM (ZT16) (Fig. 3 A ). Differently from WT mice, similar numbers of S. Typhimurium were recovered 72 h p.i. (Fig. 3 A ). Similar results were also obtained when Clock -de fi cient mice ( Clock − / − ) and their WT littermates were infected, thus con fi rming that a functional CLOCK protein is necessary for the observed effect. (Fig. S4 A ). Furthermore, a signi fi cant difference was found in the colonization of Peyer ’ s patches between WT mice infected at day and Clock ...
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... LCN2 – sensitive mutant in the salmochelin receptor IroN has a colonization defect and is outgrown by Salmonella WT in mice expressing Lcn2 , but not in mice carrying a Lcn2 mutation (11). Thus, we hypothesized that WT S. Typhimurium would have a greater colonization advantage in competition with the iroN mutant during the day, when Lcn2 is highly induced, whereas we would recover both strains at similar levels at night, when Lcn2 is expressed at lower levels. Infection of WT mice at ZT4 or ZT16 with a 1:1 mixture of WT S. Typhimurium and the iroN mutant con fi rmed this prediction. Salmonella WT outcompeted the iroN mutant in the colon content collected 72 h p.i. from mice infected during the day (Fig. 4 E ). In contrast, S. Typhimurium WT had no signi fi cant advantage over the iroN mutant at night (Fig. 4 E ), when Lcn2 was induced at lower levels. These results further suggest that circadian expression of Lcn2 , and possibly other antimicrobial peptides, in fl uences Salmonella colonization and its competitive advantage over susceptible microbes. Although in lower organisms a direct connection between the circadian system and susceptibility to infection has been established (30 – 31), this link has remained elusive in mammals. Here we show that the host response to Salmonella infection changes between day and night, corresponding to rest and active phase in mice, and that this differential response is driven by the circadian clock. Immedi- ately after infection, the in fl ammatory response increases by following an oscillatory circadian course, with higher response during the day and reduction at night. This effect is a consequence of the clock-controlled expression of many genes whose products play a fundamental role in host defense against infection. Among these, genes encoding antimicrobial peptides display a robust circadian oscillation during infection, resulting in the modulation of Salmonella colonization and its competition with susceptible microorganisms for a niche in the in fl amed gut. Although Salmonella is resistant to several antimicrobial responses, including LCN2 and REG3 γ , commensal microbes are more susceptible. Therefore, circadian regulation of antimicrobial proteins may be important to control the overgrowth of the microbiota and prevent infection from susceptible microorganisms. Previous reports have shed light on the effect that ablation of clock genes has on speci fi c immune parameters (32). In fl ies, two circadian mutants are more sensitive to some bacterial pathogens than WT organisms (33). Furthermore, Per2 -de fi cient mice are more resistant to LPS-induced endotoxic shock than WT mice (34), and disruption of the circadian clockwork by targeting Bmal1 or Nr1d1 removed the circadian gating of the endotoxin-induced cytokine response, both in vitro and in vivo (17). Our results show that Clock mutant mice have an altered timing of reaction following Salmonella infection, as well as a signi fi cant reduction in the expression of proin fl ammatory genes. Although a circadian clock-independent function of clock genes cannot be formally excluded, recent fi ndings also suggest that al- teration of circadian gene expression is likely to be responsible for the phenotype of Clock mutant mice (17, 35). In our experiments, rhythmicity of circadian and metabolic genes is suppressed or attenuated at the site of infection, supporting the concept of intimate bidirectional communication between the circadian and immune systems (36, 37). Our hypothesis is that immune factors contribute to the daily coordination of the circadian system, but powerful immunological challenges send signals to disrupt this regulation, possibly by uncoupling some of the circadian outputs, leading to reduction of the amplitude of the oscillations. Con- versely, the circadian system dictates the right timing for the host response to infection by regulating components of the immune system both at the level of individual cells and at the systemic level, through autonomic and endocrine outputs (18). The circadian system modulates the activity of several transcription factors that are important regulators of immune functions, including HIF1- α , STAT1, STAT3, and NF- κ B (38 – 40). We were able to con fi rm that these factors, together with many more, create the main transcriptional regulatory networks during infection in our genomic pro fi ling analysis. The results of our analysis point to connections of the circadian clock to other functional systems, including metabolic and immune, during infection and will be instrumental for future studies focused on elucidating these mechanisms. Animals. All experiments were approved by the Institutional An- imal Care and Use Committee at the University of California, Irvine. For the generation of Clock mutant mice, see ref. 1. Clock de fi cient (Clock − / − ) mice were a gift from S. Reppert (University of Massachusetts Medical School, Worchester, MA) (2). For experiments in Fig. S3, we used C57BL/6J wild-type (WT) mice of 8 – 12 wk of age. All experiments were conducted a minimum of two times with similar results, even though some degree of variability between WT mice was found, depending on mice housing conditions (different vivaria, different degree of sterility of cages, and food). For the microarray study, Clock mutant mice and their WT littermates were used. Bacterial Strains and Culture Conditions. We used Salmonella enterica serovar Typhimurium ( S. Typhimurium) IR715, a fully vir- ulent, nalidixic acid-resistant derivative of isolate ATCC14028 (3). Mutants in invA , fl iC fl jB , msbB , and iroN were described (4, 5). All strains were cultured aerobically in Luria Bertani (LB) broth at 37 °C, with the exception of the msbB mutant, which was grown in low-salt LB medium (4). In the cfu counts, outliers were detected with the Grubbs outlier test. All outliers with α con fi dence level of ≤ 0.01 were removed from the counts. RNA Extraction and Real-Time PCR. For gene expression analysis by real-time PCR, total RNA was extracted with TRIzol Reagent and processed according to the instructions of the manufacturer. Next, 2 μ g of RNA from each sample was retrotranscribed (Su- perScript II Reverse Transcriptase; Invitrogen), and fourfold di- luted cDNA was used for each real time reaction. For 20 μ L of PCR, 50 ng of cDNA was mixed with primers to a fi nal concentration of 150 nM and 4 μ L of RT 2 SYBR Green Fluor Fast master mix (QIAGEN). The reaction was fi rst incubated at 95 °C for 3 min, followed by 40 cycles at 95 °C for 30 s and 60 °C for 1 min. Each quantitative real-time PCR was performed by using the Chromo4 real time detection system (Bio-Rad). All values are relative to those of Gapdh or β -actin mRNA levels at each time point. A list of the real-time primers used in this study is provided in Table S1. Analysis was performed in Fig. 4 B by calculating the geometric means of fold changes in gene expression of infected compared with uninfected mice killed at the same time during the day. In Fig. 4 C , values in uninfected WT day were set to 1. Bars represent mean ± SEM ( n = 7 – 14 for infected mice; n = 3 – 5 for uninfected mice). Bone Marrow-Derived Macrophage Preparations. Three Clock mutant mice and three isogenic WT mice were killed between zeitgeber time (ZT) 4 and ZT10, and bone marrow was extracted from femurs of each animal. Differentiation of macrophages was obtained by culturing the bone marrow in RPMI (GIBCO) supplemented with 10% (vol/vol) L -929 conditioned medium, 10% (vol/vol) FBS, 2 mM glutamine, and antibiotics. After 7 d, cells were counted and plated for the experiment. Bone marrow-derived macrophages (BMDMs) were plated for the experiment and treated with lipopolysaccharide (LPS) at a concentration of 1 μ g/mL (Ultrapure LPS; Invivogen) or infected with S. Typhimurium by using a gentamicin protection assay as described (4). Cells were harvested, and samples were processed for RNA extraction. For circadian experiments using the dexamethasone (DEX) method, BMDMs were synchronized by treatment with 100 nM DEX (Sigma) for 2 h. For circadian experiments using the serum shock method, after 2 h of serum shock with medium containing 50% (vol/vol) horse serum, cells were in- cubated with serum-free medium for the indicated time. LPS treatment (1 μ g/mL) was performed at the indicated time points. Enzyme-Linked ImmunoSorbent Assay. Supernatant from BMDMs was collected 24 h poststimulation with LPS or after in vitro infection with S. Typhimurium WT or mutants. Secretion of TNF- α , IL-6, and -1 β was detected by using commercially available kits (eBioscience). Histopathology. Tissue samples were fi xed in formalin, processed according to standard procedures for paraf fi n embedding, sectioned at 5 μ m, and stained with hematoxylin and eosin. The pathology score of cecal samples was determined by blinded examinations of cecal sections from a board-certi fi ed pathologist. Each section was evaluated for the presence of neutrophils, mononuclear in fi ltrate, submucosal edema, surface erosions, in fl ammatory exudates, and cryptitis. In fl ammatory changes were scored from 0 to 4 according to the following scale: 0, none; 1, low; 2, moderate; 3, high; 4, ex- treme. The in fl ammation score was calculated by adding up all of the scores obtained for each parameter and interpreted as follows: 0 – 2, within normal limits; 3 – 5, mild; 6 – 8, moderate; 8 + , severe. Gene Expression Pro fi ling and Statistical Analysis. RNA extracted from mice cecum was isolated by using the RNeasy Mini Cleanup kit (Qiagen). Ampli fi cation and labeling of mRNA, hybridization to Mouse Gene 1.0 ST GeneChip arrays (Affymetrix), staining, and scanning were performed according to protocols in the Affymetrix Gene Expression Analysis Technical Manual. Analyses of microarray data were performed by using BRB Array tools (Version 4.1.0 Stable Release) developed by Richard Simon and the ...
Context 5
... a pathogen is not known. Here we used the mouse colitis model to determine how the circadian clock regulates the host response during acute Salmonella infection. Differential Day – Night Response to Salmonella Infection. To determine whether the circadian clock regulates the host response to infection with S. Typhimurium, we infected wild-type (WT) mice by oral gavage either at 10:00 AM (day, early rest phase; zeitgeber time 4, ZT4) or at 10:00 PM (night, early active phase; ZT16). At 48, 60, 72, and 78 h postinfection (p.i.), mice were killed, and tissue samples collected for bacteriology, histopathology, and gene expression analyses (Fig. 1). We observed that S. Typhimurium colonization signi fi cantly changed with time of infection, espe- cially at later time points. Notably, S. Typhimurium numbers were signi fi cantly increased in the colon content of mice infected at 10:00 AM in comparison with 10:00 PM at both 72 and 78 h p.i. (Fig. 1 A ); colonization of Peyer ’ s patches and spleen was also signi fi cantly higher in mice infected during the day (Fig. S1 A ). Next, we determined whether the degree of the host response to infection changed with the time of inoculation. Histopathology showed that ceca from mice infected during the day were on average more in fl amed than those from mice infected during the night, at 48, 72, and 78 h p.i. (Fig. 1 B and C ; Figs. S1 B and S2). In contrast, at 60 h p.i., a mild increase in in fl ammation was observed in mice infected at night, suggesting that the time of infection was not the only variable in fl uencing the in fl ammatory response. Reduced signs of cecal in fl ammation were characterized by low-grade submucosal edema and neutrophil in fl ux (Fig. 1 B and C and Figs. S1 B and S2). Major differences were also found in the levels of cryptitis and surface erosions (Fig. S1 B ), which constitute other classical signs of in fl ammation (10 – 12). To ascertain whether these differences were accompanied by changes in gene expression, we analyzed expression of proin fl ammatory cytokine Tnf α , neutrophil chemoattractant chemokine (C-X-C motif) ligand 1 ( Cxcl-1 ), and antimicrobial peptide lipocalin-2 ( Lcn-2 ) in the cecum of infected and uninfected mice and found signi fi cant differences during the course of infection (Fig. 1 D and Table S1). Consistent with our previous fi ndings (11 – 13), mice infected during the day showed increased expression of these genes compared with uninfected controls, and the expression was dependent both on time of infection and time of death (Fig. 1 D ). Genes in mice infected in the morning (ZT4) or at night (ZT16) showed maximal expression when killed during the day (48 and 72 h for mice infected at ZT4; 60 h for mice infected at ZT16). This fi nding was particularly notable for Tnf α because its expression was increased at both 48 and 72 h in mice infected at ZT4 (ZT4 death) relative to those infected at ZT16 (ZT16 death), and the opposite trend was observed at 60 h (ZT16 death for mice infected at ZT4; ZT4 for mice infected at ZT16). In line with this observation is the absence of signi fi cant changes in expression in mice killed 78 h after infection, corresponding to ZT10 for mice infected at ZT4 and ZT22 for mice infected at ZT16 (Fig. 1 D ). Additionally, the overall expression levels of the three proin fl ammatory genes analyzed were reduced in mice infected at ZT16 (yellow and gray graphs of Fig. 1 D ), consistently with the differences observed in colonization level and pathology score. To establish whether circadian transcription follows a normal pro fi le during infection we analyzed the expression of the clock gene period 2 ( Per2 ). Per2 oscillation in uninfected mice demonstrated proper synchronization (Fig. 1 D ). Notably, Per2 expression was progressively down-regulated after infection (Fig. 1 D ). These results suggest that a circadian mechanism may regulate components of the innate immune response to acute Salmonella infection, also revealing a profound repression of circadian components during infection. Salmonella -Infected Bone Marrow-Derived Macrophages. Our fi nding that the in fl ammatory response to S. Typhimurium infection is time-of-day – dependent in vivo prompted us to determine whether we could reproduce similar results in vitro in a less complex system. As detailed earlier, macrophages are involved in the fi rst response to S. Typhimurium infection. Moreover, macrophages have an ef fi cient clock machinery, and LPS-activated pathways in these cells are under tight circadian regulation at multiple levels (3, 4). However, bone marrow-derived macrophages (BMDMs) do not synchronously oscillate after 1 wk of differentiation in vitro (Fig. S3 A ), and LPS stimulation at different circadian times leads to similar levels of Il-6 expression in these asynchronous cultures (Fig. S3 B ). We therefore tested whether BMDMs could be synchronized by common methods such as dexamethasone or high-serum treatments (Fig. S3 C and D ). Oscillation of circadian genes Per2 (Fig. S3 C and D ), cryptochrome 1 ( Cry1 ) and brain and muscle ARNT-like protein 1 ( Bmal1 ) (Fig. S3 C ) con fi rmed entrainment of these cells. To better evaluate the contribution of the circadian system to the expression of proin fl ammatory genes, we added LPS (1 μ g/mL) to synchronized macrophages at different times of their circadian cycle and followed the expression pro fi le of Il-6 (Fig. S3 D ). As expected, we obtained different curves of expression depending on the time of LPS administration, with minimal induction for administration at T18 or T30 and major induction at T12 or T24, where T0 is the time when synchronization began. Notably, expression levels appear to be dependent both on the time of treatment and on the time of collection, as suggested by our in vivo data. We next ascertained whether disruption of the circadian clock could affect the function of some central component of the LPS response. Circadian gene expression in mice carrying a mutation of the master circadian regulator Clock [ Clock mutant or clock/ clock mice (19)] is both phase-shifted by 8 h as well as reduced compared with WT littermates (20). Clock mutant mice also fail to rhythmically express a number of immunoregulatory genes in the liver (21). Therefore, we followed the timing of expression of different cytokines after LPS stimulation of BMDMs isolated from either WT or Clock mutant mice (Fig. 2). Remarkably, we observed an overall reduction in the fold induction of the proin fl ammatory genes Il-6 , Il-1 β , Tnf α , Cxcl-1 , Ifn- β , and Che- mokine (C-C motif) ligand 2 ( Ccl2 ) in response to LPS stimulation in BMDMs isolated from Clock mutant mice compared with WT mice (Fig. 2 A ); BMDMs isolated from Clock mutant mice also exhibited reduced secretion of IL-6 and TNF- α after 24 h of LPS stimulation (Fig. 2 B ). A comparable reduction in the levels of IL-6 in the supernatant of BMDMs isolated from Clock mutant mice, compared with WT mice, was obtained after infection with S. Typhimurium (Fig. 2 C ). Because the induction of IL-6 is largely dependent on LPS stimulation of the TLR4 pathway, we also infected BMDMs with an isogenic S. Typhimurium strain carrying a mutation in the lipid A acylation pathway ( msbB mutant), which impairs signaling through TLR4 (11). As predicted, secretion of IL-6 was reduced in BMDMs infected with the msbB mutant compared with infection with WT S. Typhimurium. In contrast, both S. Typhimurium WT and the msbB mutant elicited similar low levels of secretion of IL-6 (Fig. 2 C ) in BMDMs isolated from Clock mutant mice, indicating that TLR4 signaling in response to S. Typhimurium was not induced in the absence of a functional clock. Next we sought to in- vestigate whether the secretion of the proin fl ammatory cytokine IL-1 β was also impaired in Clock mutant mice. IL-1 β is directly induced by TNF- α , causing its expression to peak later than Tnf α , and mature IL-1 β secretion requires both TLR4 and NLR signaling (22). Upon S. Typhimurium infection of BMDMs, LPS activation of TLR4 induces pro – IL-1 β expression, which is followed by NLR-mediated signaling through ice protease-activating factor/NLR family CARD domain-containing protein 4 (Ipaf/ Nlrc4) in response to Salmonella T3SS-1 secretion of fl agellin into the cytosol (22). As expected from these earlier studies, S. Typhimurium strains carrying either a mutation in msbB (unable to signal through TLR4), a mutation in both fl agellin genes ( fl iC fl jB mutant, unable to signal through Ipaf/Nlrc4), or a mutation in T3SS-1 ( invA mutant, unable to signal through Ipaf/Nlrc4) induced secretion of lower levels of IL-1 β compared with S. Typhimurium WT (Fig. 2 D ) in BMDMs from WT mice. In contrast, infection of BMDMs from Clock mutant mice with S. Typhimurium WT resulted in a marked reduction of IL-1 β secretion. Moreover, all S. Typhimurium mutants elicited similar low levels of IL-1 β in Clock mutant BMDMs. Col- lectively, we observed that secretion of proin fl ammatory cytokines in Clock mutant BMDMs was particularly low and comparable to levels elicited by S. Typhimurium mutants designed to evade activation of particular components of the in fl ammatory response. Thus, an intact circadian machinery appears to be required for the induction of proin fl ammatory cytokines in vitro in response to Salmonella infection. Mice. Because the circadian clock is necessary for a robust proin- fl ammatory response in isolated macrophages, we explored its in- volvement in the host response to Salmonella in vivo. We infected Clock mutant mice at 10:00 AM (ZT4) or 10:00 PM (ZT16) (Fig. 3 A ). Differently from WT mice, similar numbers of S. Typhimurium were recovered 72 h p.i. (Fig. 3 A ). Similar results were also obtained when Clock -de fi cient mice ( Clock − / − ) and their WT littermates were infected, thus con fi rming that a functional CLOCK protein is necessary for the observed ...
Context 6
... systems have been recently established (17, 18). Nonetheless, whether the circadian clock also in fl uences the host response to a pathogen is not known. Here we used the mouse colitis model to determine how the circadian clock regulates the host response during acute Salmonella infection. Differential Day – Night Response to Salmonella Infection. To determine whether the circadian clock regulates the host response to infection with S. Typhimurium, we infected wild-type (WT) mice by oral gavage either at 10:00 AM (day, early rest phase; zeitgeber time 4, ZT4) or at 10:00 PM (night, early active phase; ZT16). At 48, 60, 72, and 78 h postinfection (p.i.), mice were killed, and tissue samples collected for bacteriology, histopathology, and gene expression analyses (Fig. 1). We observed that S. Typhimurium colonization signi fi cantly changed with time of infection, espe- cially at later time points. Notably, S. Typhimurium numbers were signi fi cantly increased in the colon content of mice infected at 10:00 AM in comparison with 10:00 PM at both 72 and 78 h p.i. (Fig. 1 A ); colonization of Peyer ’ s patches and spleen was also signi fi cantly higher in mice infected during the day (Fig. S1 A ). Next, we determined whether the degree of the host response to infection changed with the time of inoculation. Histopathology showed that ceca from mice infected during the day were on average more in fl amed than those from mice infected during the night, at 48, 72, and 78 h p.i. (Fig. 1 B and C ; Figs. S1 B and S2). In contrast, at 60 h p.i., a mild increase in in fl ammation was observed in mice infected at night, suggesting that the time of infection was not the only variable in fl uencing the in fl ammatory response. Reduced signs of cecal in fl ammation were characterized by low-grade submucosal edema and neutrophil in fl ux (Fig. 1 B and C and Figs. S1 B and S2). Major differences were also found in the levels of cryptitis and surface erosions (Fig. S1 B ), which constitute other classical signs of in fl ammation (10 – 12). To ascertain whether these differences were accompanied by changes in gene expression, we analyzed expression of proin fl ammatory cytokine Tnf α , neutrophil chemoattractant chemokine (C-X-C motif) ligand 1 ( Cxcl-1 ), and antimicrobial peptide lipocalin-2 ( Lcn-2 ) in the cecum of infected and uninfected mice and found signi fi cant differences during the course of infection (Fig. 1 D and Table S1). Consistent with our previous fi ndings (11 – 13), mice infected during the day showed increased expression of these genes compared with uninfected controls, and the expression was dependent both on time of infection and time of death (Fig. 1 D ). Genes in mice infected in the morning (ZT4) or at night (ZT16) showed maximal expression when killed during the day (48 and 72 h for mice infected at ZT4; 60 h for mice infected at ZT16). This fi nding was particularly notable for Tnf α because its expression was increased at both 48 and 72 h in mice infected at ZT4 (ZT4 death) relative to those infected at ZT16 (ZT16 death), and the opposite trend was observed at 60 h (ZT16 death for mice infected at ZT4; ZT4 for mice infected at ZT16). In line with this observation is the absence of signi fi cant changes in expression in mice killed 78 h after infection, corresponding to ZT10 for mice infected at ZT4 and ZT22 for mice infected at ZT16 (Fig. 1 D ). Additionally, the overall expression levels of the three proin fl ammatory genes analyzed were reduced in mice infected at ZT16 (yellow and gray graphs of Fig. 1 D ), consistently with the differences observed in colonization level and pathology score. To establish whether circadian transcription follows a normal pro fi le during infection we analyzed the expression of the clock gene period 2 ( Per2 ). Per2 oscillation in uninfected mice demonstrated proper synchronization (Fig. 1 D ). Notably, Per2 expression was progressively down-regulated after infection (Fig. 1 D ). These results suggest that a circadian mechanism may regulate components of the innate immune response to acute Salmonella infection, also revealing a profound repression of circadian components during infection. Salmonella -Infected Bone Marrow-Derived Macrophages. Our fi nding that the in fl ammatory response to S. Typhimurium infection is time-of-day – dependent in vivo prompted us to determine whether we could reproduce similar results in vitro in a less complex system. As detailed earlier, macrophages are involved in the fi rst response to S. Typhimurium infection. Moreover, macrophages have an ef fi cient clock machinery, and LPS-activated pathways in these cells are under tight circadian regulation at multiple levels (3, 4). However, bone marrow-derived macrophages (BMDMs) do not synchronously oscillate after 1 wk of differentiation in vitro (Fig. S3 A ), and LPS stimulation at different circadian times leads to similar levels of Il-6 expression in these asynchronous cultures (Fig. S3 B ). We therefore tested whether BMDMs could be synchronized by common methods such as dexamethasone or high-serum treatments (Fig. S3 C and D ). Oscillation of circadian genes Per2 (Fig. S3 C and D ), cryptochrome 1 ( Cry1 ) and brain and muscle ARNT-like protein 1 ( Bmal1 ) (Fig. S3 C ) con fi rmed entrainment of these cells. To better evaluate the contribution of the circadian system to the expression of proin fl ammatory genes, we added LPS (1 μ g/mL) to synchronized macrophages at different times of their circadian cycle and followed the expression pro fi le of Il-6 (Fig. S3 D ). As expected, we obtained different curves of expression depending on the time of LPS administration, with minimal induction for administration at T18 or T30 and major induction at T12 or T24, where T0 is the time when synchronization began. Notably, expression levels appear to be dependent both on the time of treatment and on the time of collection, as suggested by our in vivo data. We next ascertained whether disruption of the circadian clock could affect the function of some central component of the LPS response. Circadian gene expression in mice carrying a mutation of the master circadian regulator Clock [ Clock mutant or clock/ clock mice (19)] is both phase-shifted by 8 h as well as reduced compared with WT littermates (20). Clock mutant mice also fail to rhythmically express a number of immunoregulatory genes in the liver (21). Therefore, we followed the timing of expression of different cytokines after LPS stimulation of BMDMs isolated from either WT or Clock mutant mice (Fig. 2). Remarkably, we observed an overall reduction in the fold induction of the proin fl ammatory genes Il-6 , Il-1 β , Tnf α , Cxcl-1 , Ifn- β , and Che- mokine (C-C motif) ligand 2 ( Ccl2 ) in response to LPS stimulation in BMDMs isolated from Clock mutant mice compared with WT mice (Fig. 2 A ); BMDMs isolated from Clock mutant mice also exhibited reduced secretion of IL-6 and TNF- α after 24 h of LPS stimulation (Fig. 2 B ). A comparable reduction in the levels of IL-6 in the supernatant of BMDMs isolated from Clock mutant mice, compared with WT mice, was obtained after infection with S. Typhimurium (Fig. 2 C ). Because the induction of IL-6 is largely dependent on LPS stimulation of the TLR4 pathway, we also infected BMDMs with an isogenic S. Typhimurium strain carrying a mutation in the lipid A acylation pathway ( msbB mutant), which impairs signaling through TLR4 (11). As predicted, secretion of IL-6 was reduced in BMDMs infected with the msbB mutant compared with infection with WT S. Typhimurium. In contrast, both S. Typhimurium WT and the msbB mutant elicited similar low levels of secretion of IL-6 (Fig. 2 C ) in BMDMs isolated from Clock mutant mice, indicating that TLR4 signaling in response to S. Typhimurium was not induced in the absence of a functional clock. Next we sought to in- vestigate whether the secretion of the proin fl ammatory cytokine IL-1 β was also impaired in Clock mutant mice. IL-1 β is directly induced by TNF- α , causing its expression to peak later than Tnf α , and mature IL-1 β secretion requires both TLR4 and NLR signaling (22). Upon S. Typhimurium infection of BMDMs, LPS activation of TLR4 induces pro – IL-1 β expression, which is followed by NLR-mediated signaling through ice protease-activating factor/NLR family CARD domain-containing protein 4 (Ipaf/ Nlrc4) in response to Salmonella T3SS-1 secretion of fl agellin into the cytosol (22). As expected from these earlier studies, S. Typhimurium strains carrying either a mutation in msbB (unable to signal through TLR4), a mutation in both fl agellin genes ( fl iC fl jB mutant, unable to signal through Ipaf/Nlrc4), or a mutation in T3SS-1 ( invA mutant, unable to signal through Ipaf/Nlrc4) induced secretion of lower levels of IL-1 β compared with S. Typhimurium WT (Fig. 2 D ) in BMDMs from WT mice. In contrast, infection of BMDMs from Clock mutant mice with S. Typhimurium WT resulted in a marked reduction of IL-1 β secretion. Moreover, all S. Typhimurium mutants elicited similar low levels of IL-1 β in Clock mutant BMDMs. Col- lectively, we observed that secretion of proin fl ammatory cytokines in Clock mutant BMDMs was particularly low and comparable to levels elicited by S. Typhimurium mutants designed to evade activation of particular components of the in fl ammatory response. Thus, an intact circadian machinery appears to be required for the induction of proin fl ammatory cytokines in vitro in response to Salmonella infection. Mice. Because the circadian clock is necessary for a robust proin- fl ammatory response in isolated macrophages, we explored its in- volvement in the host response to Salmonella in vivo. We infected Clock mutant mice at 10:00 AM (ZT4) or 10:00 PM (ZT16) (Fig. 3 A ). Differently from WT mice, similar numbers of S. Typhimurium were recovered 72 h p.i. (Fig. 3 A ). Similar results were also obtained when Clock -de fi cient mice ( ...
Context 7
... time during the 24-h light – dark (LD) cycle (15, 16). Furthermore, direct molecular links between the circadian and innate immune systems have been recently established (17, 18). Nonetheless, whether the circadian clock also in fl uences the host response to a pathogen is not known. Here we used the mouse colitis model to determine how the circadian clock regulates the host response during acute Salmonella infection. Differential Day – Night Response to Salmonella Infection. To determine whether the circadian clock regulates the host response to infection with S. Typhimurium, we infected wild-type (WT) mice by oral gavage either at 10:00 AM (day, early rest phase; zeitgeber time 4, ZT4) or at 10:00 PM (night, early active phase; ZT16). At 48, 60, 72, and 78 h postinfection (p.i.), mice were killed, and tissue samples collected for bacteriology, histopathology, and gene expression analyses (Fig. 1). We observed that S. Typhimurium colonization signi fi cantly changed with time of infection, espe- cially at later time points. Notably, S. Typhimurium numbers were signi fi cantly increased in the colon content of mice infected at 10:00 AM in comparison with 10:00 PM at both 72 and 78 h p.i. (Fig. 1 A ); colonization of Peyer ’ s patches and spleen was also signi fi cantly higher in mice infected during the day (Fig. S1 A ). Next, we determined whether the degree of the host response to infection changed with the time of inoculation. Histopathology showed that ceca from mice infected during the day were on average more in fl amed than those from mice infected during the night, at 48, 72, and 78 h p.i. (Fig. 1 B and C ; Figs. S1 B and S2). In contrast, at 60 h p.i., a mild increase in in fl ammation was observed in mice infected at night, suggesting that the time of infection was not the only variable in fl uencing the in fl ammatory response. Reduced signs of cecal in fl ammation were characterized by low-grade submucosal edema and neutrophil in fl ux (Fig. 1 B and C and Figs. S1 B and S2). Major differences were also found in the levels of cryptitis and surface erosions (Fig. S1 B ), which constitute other classical signs of in fl ammation (10 – 12). To ascertain whether these differences were accompanied by changes in gene expression, we analyzed expression of proin fl ammatory cytokine Tnf α , neutrophil chemoattractant chemokine (C-X-C motif) ligand 1 ( Cxcl-1 ), and antimicrobial peptide lipocalin-2 ( Lcn-2 ) in the cecum of infected and uninfected mice and found signi fi cant differences during the course of infection (Fig. 1 D and Table S1). Consistent with our previous fi ndings (11 – 13), mice infected during the day showed increased expression of these genes compared with uninfected controls, and the expression was dependent both on time of infection and time of death (Fig. 1 D ). Genes in mice infected in the morning (ZT4) or at night (ZT16) showed maximal expression when killed during the day (48 and 72 h for mice infected at ZT4; 60 h for mice infected at ZT16). This fi nding was particularly notable for Tnf α because its expression was increased at both 48 and 72 h in mice infected at ZT4 (ZT4 death) relative to those infected at ZT16 (ZT16 death), and the opposite trend was observed at 60 h (ZT16 death for mice infected at ZT4; ZT4 for mice infected at ZT16). In line with this observation is the absence of signi fi cant changes in expression in mice killed 78 h after infection, corresponding to ZT10 for mice infected at ZT4 and ZT22 for mice infected at ZT16 (Fig. 1 D ). Additionally, the overall expression levels of the three proin fl ammatory genes analyzed were reduced in mice infected at ZT16 (yellow and gray graphs of Fig. 1 D ), consistently with the differences observed in colonization level and pathology score. To establish whether circadian transcription follows a normal pro fi le during infection we analyzed the expression of the clock gene period 2 ( Per2 ). Per2 oscillation in uninfected mice demonstrated proper synchronization (Fig. 1 D ). Notably, Per2 expression was progressively down-regulated after infection (Fig. 1 D ). These results suggest that a circadian mechanism may regulate components of the innate immune response to acute Salmonella infection, also revealing a profound repression of circadian components during infection. Salmonella -Infected Bone Marrow-Derived Macrophages. Our fi nding that the in fl ammatory response to S. Typhimurium infection is time-of-day – dependent in vivo prompted us to determine whether we could reproduce similar results in vitro in a less complex system. As detailed earlier, macrophages are involved in the fi rst response to S. Typhimurium infection. Moreover, macrophages have an ef fi cient clock machinery, and LPS-activated pathways in these cells are under tight circadian regulation at multiple levels (3, 4). However, bone marrow-derived macrophages (BMDMs) do not synchronously oscillate after 1 wk of differentiation in vitro (Fig. S3 A ), and LPS stimulation at different circadian times leads to similar levels of Il-6 expression in these asynchronous cultures (Fig. S3 B ). We therefore tested whether BMDMs could be synchronized by common methods such as dexamethasone or high-serum treatments (Fig. S3 C and D ). Oscillation of circadian genes Per2 (Fig. S3 C and D ), cryptochrome 1 ( Cry1 ) and brain and muscle ARNT-like protein 1 ( Bmal1 ) (Fig. S3 C ) con fi rmed entrainment of these cells. To better evaluate the contribution of the circadian system to the expression of proin fl ammatory genes, we added LPS (1 μ g/mL) to synchronized macrophages at different times of their circadian cycle and followed the expression pro fi le of Il-6 (Fig. S3 D ). As expected, we obtained different curves of expression depending on the time of LPS administration, with minimal induction for administration at T18 or T30 and major induction at T12 or T24, where T0 is the time when synchronization began. Notably, expression levels appear to be dependent both on the time of treatment and on the time of collection, as suggested by our in vivo data. We next ascertained whether disruption of the circadian clock could affect the function of some central component of the LPS response. Circadian gene expression in mice carrying a mutation of the master circadian regulator Clock [ Clock mutant or clock/ clock mice (19)] is both phase-shifted by 8 h as well as reduced compared with WT littermates (20). Clock mutant mice also fail to rhythmically express a number of immunoregulatory genes in the liver (21). Therefore, we followed the timing of expression of different cytokines after LPS stimulation of BMDMs isolated from either WT or Clock mutant mice (Fig. 2). Remarkably, we observed an overall reduction in the fold induction of the proin fl ammatory genes Il-6 , Il-1 β , Tnf α , Cxcl-1 , Ifn- β , and Che- mokine (C-C motif) ligand 2 ( Ccl2 ) in response to LPS stimulation in BMDMs isolated from Clock mutant mice compared with WT mice (Fig. 2 A ); BMDMs isolated from Clock mutant mice also exhibited reduced secretion of IL-6 and TNF- α after 24 h of LPS stimulation (Fig. 2 B ). A comparable reduction in the levels of IL-6 in the supernatant of BMDMs isolated from Clock mutant mice, compared with WT mice, was obtained after infection with S. Typhimurium (Fig. 2 C ). Because the induction of IL-6 is largely dependent on LPS stimulation of the TLR4 pathway, we also infected BMDMs with an isogenic S. Typhimurium strain carrying a mutation in the lipid A acylation pathway ( msbB mutant), which impairs signaling through TLR4 (11). As predicted, secretion of IL-6 was reduced in BMDMs infected with the msbB mutant compared with infection with WT S. Typhimurium. In contrast, both S. Typhimurium WT and the msbB mutant elicited similar low levels of secretion of IL-6 (Fig. 2 C ) in BMDMs isolated from Clock mutant mice, indicating that TLR4 signaling in response to S. Typhimurium was not induced in the absence of a functional clock. Next we sought to in- vestigate whether the secretion of the proin fl ammatory cytokine IL-1 β was also impaired in Clock mutant mice. IL-1 β is directly induced by TNF- α , causing its expression to peak later than Tnf α , and mature IL-1 β secretion requires both TLR4 and NLR signaling (22). Upon S. Typhimurium infection of BMDMs, LPS activation of TLR4 induces pro – IL-1 β expression, which is followed by NLR-mediated signaling through ice protease-activating factor/NLR family CARD domain-containing protein 4 (Ipaf/ Nlrc4) in response to Salmonella T3SS-1 secretion of fl agellin into the cytosol (22). As expected from these earlier studies, S. Typhimurium strains carrying either a mutation in msbB (unable to signal through TLR4), a mutation in both fl agellin genes ( fl iC fl jB mutant, unable to signal through Ipaf/Nlrc4), or a mutation in T3SS-1 ( invA mutant, unable to signal through Ipaf/Nlrc4) induced secretion of lower levels of IL-1 β compared with S. Typhimurium WT (Fig. 2 D ) in BMDMs from WT mice. In contrast, infection of BMDMs from Clock mutant mice with S. Typhimurium WT resulted in a marked reduction of IL-1 β secretion. Moreover, all S. Typhimurium mutants elicited similar low levels of IL-1 β in Clock mutant BMDMs. Col- lectively, we observed that secretion of proin fl ammatory cytokines in Clock mutant BMDMs was particularly low and comparable to levels elicited by S. Typhimurium mutants designed to evade activation of particular components of the in fl ammatory response. Thus, an intact circadian machinery appears to be required for the induction of proin fl ammatory cytokines in vitro in response to Salmonella infection. Mice. Because the circadian clock is necessary for a robust proin- fl ammatory response in isolated macrophages, we explored its in- volvement in the host response to Salmonella in vivo. We infected Clock mutant mice at 10:00 AM (ZT4) or 10:00 PM (ZT16) (Fig. 3 A ). Differently from WT mice, ...

Citations

... ↓ expression of pro-inflammatory genes Il-6, Il-1β, Tnfα, Cxcl1, Ifnβ, and Ccl2 and ↓ TNFα and IL-6 response in BMDMs [63] Clock mutant mice Salmonella infection (in vivo) Impaired rhythmicity in bacterial colonization in the gut and reduced pro-inflammatory gene expression [63] ↓ TNFα and IL-12 production in challenged peritoneal macrophages and ↓ Tlr9 expression ...
... ↓ expression of pro-inflammatory genes Il-6, Il-1β, Tnfα, Cxcl1, Ifnβ, and Ccl2 and ↓ TNFα and IL-6 response in BMDMs [63] Clock mutant mice Salmonella infection (in vivo) Impaired rhythmicity in bacterial colonization in the gut and reduced pro-inflammatory gene expression [63] ↓ TNFα and IL-12 production in challenged peritoneal macrophages and ↓ Tlr9 expression ...
... These findings were supported by reduced activation of NF-κB in response to immune challenge in mouse embryonic fibroblasts (MEFs), as well as hepatocytes of Clock-deficient mice compared to wild-type controls [49]. Similarly, reduced induction of pro-inflammatory cytokines upon LPS challenge has been observed in MEFs and BMDMs from Clock-mutant mice [63,64]. Moreover, day/night differences in inflammatory response to Salmonella infection were eliminated in the gut of Clock mutants [63]. ...
Article
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Circadian rhythms control almost all aspects of physiology and behavior, allowing temporal synchrony of these processes between each other, as well as with the external environment. In the immune system, daily rhythms of leukocyte functions can determine the strength of the immune response, thereby regulating the efficiency of defense mechanisms to cope with infections or tissue injury. The natural light/dark cycle is the prominent synchronizing agent perceived by the circadian clock, but this role of light is highly compromised by irregular working schedules and unintentional exposure to artificial light at night (ALAN). The primary concern is disrupted circadian control of important physiological processes, underlying potential links to adverse health effects. Here, we first discuss the immune consequences of genetic circadian disruption induced by mutation or deletion of specific clock genes. Next, we evaluate experimental research into the effects of disruptive light/dark regimes, particularly light-phase shifts, dim ALAN, and constant light on the innate immune mechanisms under steady state and acute inflammation, and in the pathogenesis of common lifestyle diseases. We suggest that a better understanding of the mechanisms by which circadian disruption influences immune status can be of importance in the search for strategies to minimize the negative consequences of chronodisruption on health.
... Interestingly, it has been demonstrated that the expression of Per2 is progressively decreased in the caecum of mice infected with Salmonella enterica serovar Typhimurium [S. Typhimurium, a pathogenic Gram-negative bacteria found in the intestinal lumen with its toxicity determined by lipopolysaccharide (LPS) in its outer membrane] (Bellet et al., 2013). Synchronized treatment of LPS with high-serum (serum shock) alters the amplitude of Per2 gene expression in bone marrow-derived macrophages (BMDMs) (Bellet et al., 2013), indicating that LPS could be an effective regulator of the circadian clock (Mukherji et al., 2013). ...
... Typhimurium, a pathogenic Gram-negative bacteria found in the intestinal lumen with its toxicity determined by lipopolysaccharide (LPS) in its outer membrane] (Bellet et al., 2013). Synchronized treatment of LPS with high-serum (serum shock) alters the amplitude of Per2 gene expression in bone marrow-derived macrophages (BMDMs) (Bellet et al., 2013), indicating that LPS could be an effective regulator of the circadian clock (Mukherji et al., 2013). Pathogen infection can also reprogram the expression of circadian clock genes in extraintestinal tissues. ...
... development, progression, and resolution). Mice infected with S. Typhimurium at ZT4 have higher levels of bacterial load in the colon content and higher expressions of proinflammatory cytokines (Tnfα and Cxcl1) in the caecum compared to ZT16 (Bellet et al., 2013). Additionally, circadian regulation in expression of antimicrobial proteins (e.g. ...
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Circadian rhythms are present in almost every organism and regulate multiple aspects of biological and physiological processes (e.g. metabolism, immune responses, and microbial exposure). There exists a bidirectional circadian interaction between the host and its gut microbiota, and potential circadian orchestration of both host and gut microbiota in response to invading pathogens. In this review, we summarize what is known about these intestinal microbial oscillations and the relationships between host circadian clocks and various infectious agents (bacteria, fungi, parasites, and viruses), and discuss how host circadian clocks prime the immune system to fight pathogen infections as well as the direct effects of circadian clocks on viral activity (e.g. SARS-CoV-2 entry and replication). Finally, we consider strategies employed to realign normal circadian rhythmicity for host health, such as chronotherapy, dietary intervention, good sleep hygiene, and gut microbiota-targeted therapy. We propose that targeting circadian rhythmicity may provide therapeutic opportunities for the treatment of infectious diseases.
... Our results show that the time of day that individuals are exposed Westwood et al., 2019). For example, Salmonella colonization and host inflammatory responses were higher in mice infected during the resting phase than those infected during the active phase (Bellet et al., 2013). These studies suggest that infection outcomes are often more severe when hosts are infected during the phase opposite of when they would be exposed to pathogens in nature (typically the active phase) and these differences have important implications for the conclusions drawn from epidemiological studies. ...
Article
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Biological rhythms mediate important within‐host processes such as metabolism, immunity, and behavior which are often linked to combating disease exposure. For many hosts, exposure to pathogens occurs while feeding. However, the link between feeding rhythms and infection risk is unclear because feeding behavior is tightly coupled with immune and metabolic processes which may decrease susceptibility to infection. Here, we use the Daphnia dentifera–Metschnikowia bicuspidata host–pathogen system to determine how rhythms in feeding rate and immune function mediate infection risk. The host is known to have a nocturnal circadian rhythm in feeding rate, yet we found that they do not exhibit a circadian rhythm in phenoloxidase activity. We found that the time of day when individuals are exposed to pathogens affects the probability of infection with higher infection prevalence at night, indicating that infection risk is driven by a host's circadian rhythm in feeding behavior. These results suggest that the natural circadian rhythm of the host should be considered when addressing epidemiological dynamics. The link between biological rhythms in feeding behavior and infection risk is unclear because feeding behavior is tightly coupled with immune and metabolic processes which may affect infection susceptibility. We use the Daphnia dentifera–Metschnikowia bicuspidata host–pathogen system to determine how rhythms in feeding rate and immune function mediate infection risk. Individuals are more likely to get infected during night exposures, indicating that infection risk is driven by a host's nocturnal circadian rhythm in feeding behavior.
... Stud- 25 ies have reported circadian rhythms controlling host immune response, reproduction, antibacterial host defenses, sepsis, inflammation, and cell proliferation [7,9,10,11]. As understanding of the role of circadian rhythms on infection has grown, studies conducted on pathogens such as human herpesvirus 2, influenza, Salmonella typhimurium, and Chlamydia not only suggest that circadian rhythms influence the progression of the disease, but that the time of infection seems to play an 30 essential role in overall disease outcomes [12,13,14]. ...
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Background: We have previously shown that the time of Chlamydia infection was crucial in determining the chlamydial infectivity and pathogenesis. This study aims to determine whether the time of Chlamydia infection affects the genital tract microbiome. This study analyzed mice vaginal, uterine, and ovary/oviduct microbiome with and without Chlamydia infection. The mice were infected with Chlamydia at either 10:00 am (ZT3) or 10:00 pm (ZT15). Results: The results showed that mice infected at ZT3 had higher Chlamydia infectivity than those infected at ZT15. There was more variation in the compositional complexity of the vaginal microbiome (alpha diversity) of mice infected at ZT3 than those mice infected at ZT15 throughout the infection within each treatment group, with both Shannon and Simpson diversity index values decreased over time. The analysis of samples collected four weeks post-infection showed that there were significant taxonomical differences (beta diversity) between different parts of the genital tract---vagina, uterus, and ovary/oviduct---and this difference was associated with the time of infection. Firmicutes and Proteobacteria were the most abundant phyla within the microbiome in all three genital tract regions for all the samples collected during this experiment. Additionally, Firmicutes was the dominant phylum in the uterine microbiome of ZT3 Chlamydia infected mice. Conclusion: The results show that the time of infection is associated with the microbial dynamics in the genital tract. And this association is more robust in the upper genital tract than in the vagina. This result implies that more emphasis should be placed on understanding the changes in the microbial dynamics of the upper genital tract over the course of infection.
... Besides, the microbiota rhythmicity also affected the susceptibility of a host to infection by pathogens. Bellet et al. (2013) found that the invasive capacity of Salmonella typhimurium has significant diurnal variation [90]. Meanwhile, antimicrobial peptide, which was supposed to be resistant to pathogen invasion and has fundamental implications for innate immunity, showed a rhythmic pattern [91]. ...
... Besides, the microbiota rhythmicity also affected the susceptibility of a host to infection by pathogens. Bellet et al. (2013) found that the invasive capacity of Salmonella typhimurium has significant diurnal variation [90]. Meanwhile, antimicrobial peptide, which was supposed to be resistant to pathogen invasion and has fundamental implications for innate immunity, showed a rhythmic pattern [91]. ...
... Increasing evidence suggests that certain diseases such as myocardial infarction, acute cardiovascular diseases, and stroke occur predominantly during the light phase [95,96]. More specifically, a higher pro-inflammatory response was induced by the invasion of Salmonella typhimurium during the early rest period in the mice model compared with the other times of the day [90]. In addition, the side effects of some drugs (e.g., the hepatotoxicity induced by an overdose of acetaminophen) also exhibited robust rhythmicity [89]. ...
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Full-text available
Unlike the strictly hierarchical organization in the circadian clock system, the gut microbiota rhythmicity has a more complex multilayer network of all taxonomic levels of microbial taxa and their metabolites. However, it is worth noting that the functionality of the gut microbiota rhythmicity is highly dependent on the host circadian clock and host physiological status. Here, we discussed the diurnal rhythmicity of the gut microbiota; its crucial role in host physiology, health, and metabolism; and the crosstalk between the gut microbial rhythmicity and host circadian rhythm. This knowledge lays the foundation for the development of chronotherapies targeting the gut microbiota. However, the formation mechanism, its beneficial effects on the host of gut microbial rhythmicity, and the dynamic microbial-host crosstalk are not yet clear and warrant further research.
... The circadian immune response against Salmonella Typhimurium has recently been demonstrated to be driven by oscillations in feeding behavior and the activity of the gut microbiota. Mice pretreated with streptomycin are more susceptible to oral Salmonella Typhimurium when infected early during the day (ZT4; higher bacterial count in the colon and mesenteric LNs) compared with the night (ZT16) (94). In contrast, nonantibiotic-treated mice infected with Salmonella Typhimurium orally at ZT12 exhibit an increased bacterial burden in the SI 24 hours later and suffer from significantly increased mortality compared with those infected at ZT0 (morning) (89). ...
... Bacterial infections are also affected by circadian mechanisms. As mentioned above, mice were shown to be more susceptible to oral Salmonella Typhimurium infection when gavaged in the morning compared with the evening (94). This increased susceptibility resulted in greater colonization of the colon by Salmonella Typhimurium and a greater proinflammatory response. ...
Article
The immune system is highly time-of-day dependent. Pioneering studies in the 1960s were the first to identify immune responses to be under a circadian control. Only in the last decade, however, have the molecular factors governing circadian immune rhythms been identified. These studies have revealed a highly complex picture of the interconnectivity of rhythmicity within immune cells with that of their environment. Here, we provide a global overview of the circadian immune system, focusing on recent advances in the rapidly expanding field of circadian immunology.
... 50, Web Server issue with genetic, epigenetic, tissue/organ, health, age, and environmental conditions. This circadian reprogramming is a major target of active investigations aimed at understanding how environmental conditions, such as drug treatments or diets, affect circadian oscillations and how oscillations in different cells/tissues are coordinated and interact with each other (19,(23)(24)(25)(26)(27)(28)(29)(30). The large repository of omic data available via CircadiOmics, serves as an invaluable resource to analyze the complexity of circadian mechanisms and their downstream implications. ...
Article
Full-text available
Circadian rhythms are a foundational aspect of biology. These rhythms are found at the molecular level in every cell of every living organism and they play a fundamental role in homeostasis and a variety of physiological processes. As a result, biomedical research of circadian rhythms continues to expand at a rapid pace. To support this research, CircadiOmics (http://circadiomics.igb.uci.edu/) is the largest annotated repository and analytic web server for high-throughput omic (e.g. transcriptomic, metabolomic, proteomic) circadian time series experimental data. CircadiOmics contains over 290 experiments and over 100 million individual measurements, across >20 unique tissues/organs, and 11 different species. Users are able to visualize and mine these datasets by deriving and comparing periodicity statistics for oscillating molecular species including: period, amplitude, phase, P-value and q-value. These statistics are obtained from BIO_CYCLE and JTK_CYCLE and are intuitively aggregated and displayed for comparison. CircadiOmics is the most up-to-date and cutting-edge web portal for searching and analyzing circadian omic data and is used by researchers around the world.
... Animals, plants, fungi and certain bacteria predict these environmental changes by regulating their molecular, biochemical, physiological and behavioural cycles (1). These rhythms include host immune responses to a variety of diseases and bacterial infections including those caused by Streptococcus pneumoniaethe leading causative agent of death by communicable disease, despite the availability of effective treatment and vaccines (2)(3)(4)(5)(6)(7). Using highly reliable murine infection models (8), it has been shown that infection with S. pneumoniae evidenced a strong periodicity in immune response and outcome of infection, with survival time and time-to-onset of severe bacteraemia being significantly influenced by the time-of-day of infection, and infection during the active phase often resulting in the most favourable outcome (6,(9)(10)(11). ...
Article
Full-text available
Circadian rhythms affect the progression and severity of bacterial infections including those caused by Streptococcus pneumoniae, but the mechanisms responsible for this phenomenon remain largely elusive. Following advances in our understanding of the role of replication of S. pneumoniae within splenic macrophages, we sought to investigate whether events within the spleen correlate with differential outcomes of invasive pneumococcal infection. Utilising murine invasive pneumococcal disease (IPD) models, here we report that infection during the murine active phase (zeitgeber time 15; 15h after start of light cycle, 3h after start of dark cycle) resulted in significantly faster onset of septicaemia compared to rest phase (zeitgeber time 3; 3h after start of light cycle) infection. This correlated with significantly higher pneumococcal burden within the spleen of active phase-infected mice at early time points compared to rest phase-infected mice. Whole-section confocal microscopy analysis of these spleens revealed that the number of pneumococci is significantly higher exclusively within marginal zone metallophilic macrophages (MMMs) known to allow intracellular pneumococcal replication as a prerequisite step to the onset of septicaemia. Pneumococcal clusters within MMMs were more abundant and increased in size over time in active phase-infected mice compared to those in rest phase-infected mice which decreased in size and were present in a lower percentage of MMMs. This phenomenon preceded significantly higher levels of bacteraemia alongside serum IL-6 and TNF-a concentrations in active phase-infected mice following re-seeding of pneumococci into the blood. These data greatly advance our fundamental knowledge of pneumococcal infection by linking susceptibility to invasive pneumococcal infection to variation in the propensity of MMMs to allow persistence and replication of phagocytosed bacteria. These findings also outline a somewhat rare scenario whereby the active phase of an organism's circadian cycle plays a seemingly counterproductive role in the control of invasive infection.
... The far-reaching effects of food intake on host circadian rhythms are mediated by the gut microbiota, which periodically interact with the host to regulate rhythms in both innate immunity and metabolism 10,11,13 , and potentially the gut-brain axis 9 . However, despite a long-standing appreciation for the importance of both circadian rhythms 1,4,[16][17][18][19] and the gut microbiota [20][21][22][23][24][25] for mediating host biological, ecological, and evolutionary processes, their interaction has largely been neglected in the study of natural populations. ...
... Many aspects of innate immunity are therefore downregulated during the rest phase when the gut lining becomes less permeable and the host is less likely to encounter pathogens (Fig. 3b). This leads to higher host susceptibility to pathogens during the rest phase 58 , with pathogens such as Salmonella colonising at higher abundances compared to the active phase 16 . The downregulation of innate immunity in the gut is preceded by the detachment of mucosal commensals from the mucosal layer via mechanisms which remain unclear to date, thereby triggering a reduction in the number of cytokines and AMPs secreted into the gut. ...
Preprint
Full-text available
Daily light-dark cycles shape the physiology and activity patterns of nearly all organisms. Recent evidence that gut microbial oscillations synchronise circadian rhythms in host immunity and metabolism indicate that diurnal dynamics is a crucial component of microbiome function. However, their prevalence and functional significance are rarely tested in natural populations. Here we summarize the hallmarks of gut microbiota oscillations and the mechanisms by which they synchronise rhythms in host immunity and metabolism. We discuss the consequences for diverse biological processes such as host pathogen susceptibility and seasonal switches in metabolism, and outline how the breakdown of these circadian interactions, for example during senescence and as a consequence of urbanisation, may affect wildlife infection risk and disease. Lastly, we provide practical guidelines for the measurement of microbial oscillations in wildlife, highlighting that whilst wild animals are rarely available over a 24-hour period, characterising even parts of the cycle can be informative. Light-dark cycles are an almost universal environmental cue and provide a rare opportunity to generalise gut microbial responses across species. An improved understanding of how microbial rhythms manifest in wildlife is essential to fully comprehend their ecological significance.
... The far-reaching effects of food intake on host circadian rhythms are mediated by the gut microbiota, which rhythmically interact with the host to regulate rhythms in both innate immunity and metabolism 10,11,13 . However, despite a long-standing appreciation for the importance of both circadian rhythms 1,4,[16][17][18][19] and the gut microbiota [20][21][22][23][24][25] for mediating host biological, ecological, and evolutionary processes, their interaction has largely been neglected in the study of natural populations. ...
Preprint
Daily light-dark cycles shape the activity patterns and physiology of nearly all organisms. Many biological processes undergo circadian rhythms, yet rhythms in immunity and metabolism are particularly important for the maintenance of biological homeostasis. Recent evidence that food intake and the gut bacterial microbiota synchronise system-wide circadian rhythms spanning immunity, metabolism, and behaviour point towards gut microbial oscillations being a crucial component of microbiome function. Findings from model systems suggest that gut microbial oscillations are likely widespread across species and pivotal for shaping immune and metabolic responses, yet their prevalence and functional significance are rarely tested in natural populations. Here we summarize results from experimental studies on how circadian interactions between the gut microbiota and the host act to synchronise rhythms in host metabolism and immunity. We outline how these circadian interactions are likely to mediate diverse biological processes, including host pathogen susceptibility and seasonal switches in metabolism, and discuss how the breakdown of these interactions, for example during senescence and urbanisation, can lead to dysbiosis and declines in health. Lastly, we provide practical guidelines for the measurement of microbial oscillations in wildlife, highlighting that whilst wild animals are rarely available over a 24-hour period, characterising even parts of the cycle can be informative. Light-dark cycles are an almost universal environmental cue that provide a rare opportunity to generalise gut microbial responses across species, yet to fully appreciate their ecological relevance an understanding of how microbial rhythms manifest in wildlife is essential. THIS IS A PREPRINT ACCESSIBLE HERE: https://www.authorea.com/doi/full/10.22541/au.165244698.86280644/v1