For Whom the Clock Ticks: Clinical Chronobiology for Infectious Diseases

It is well-established that the immune response varies according to circadian rhythms. These variations concern innate and adaptive immune responses at both quantitative and qualitative levels. At the quantitative level, the circulating number of hematopoietic stem and progenitor cells, and most mature leukocytes increases during the resting phase for rodents and the night for humans (). The migration of immune cells to the tissues, a major phase of anti-microbial response, occurs preferentially during the active phase. This response also involves neuro-mediators released locally by sympathetic nerves, thus underlying the role of central pacemaker in addition to peripheral clocks (). It has recently been shown that neutrophil traffic is regulated through a timer program. The deletion of aryl hydrocarbon receptor nuclear translocator-like protein 1 () andgenes prevents diurnal rhythms, called neutrophil aging (). The homing of circulating lymphocytes into lymph nodes and their egress into efferent lymphatic vessels obey to a rhythmic process. The egress of cells is based on circadian variations of CCR7 production and that of its ligand CCL21 in both T and B lymphocytes (). ^{21 22 23 24} Arntl Cxcr2

The immune functions are modulated by circadian control. Regarding the innate immune system, functions such as particle uptake and release of oxygen derivatives and cytokines exhibit circadian rhythms (,). Regarding the adaptive immunity, there is evidence that the functions of T and B lymphocytes also exhibit circadian rhythms. Indeed, the production of antibodies in response to thymo-dependent and thymo-independent antigens oscillates with melatonin rhythm in mice; melatonin suppression is associated with increased levels of specific antibodies, which are corrected by the addition of melatonin and light (). Similarly, immunoglobulin (Ig)-E levels and IgE-mediated allergic responses are regulated by molecular clock in mice () and human mast cells; similar results are obtained in human eosinophils activated with N-formyl-methionly-leucyl-phenylalanine, a canonical chemotactic peptide (). ^{25 26 27 28 29}

In mice, the peak of salivary IgA levels occurs during the night; this response seems to be under the control of a central pacemaker (). The lessons from mice invalidated for clock genes underline the role of these genes in the ontogeny of B lymphocytes and plasma cells () The functions of T lymphocytes are also controlled in a circadian manner. The proliferative response of T lymphocytes to mitogens is strongly rhythmic and is impaired when clock genes are mutated (). In addition, the clock genes are involved in the differentiation of Th17 cells. The Th17 cell development is suppressed by nuclear factor, interleulin-3 regulated (NFIL3), a circadian-regulated transcription factor;and related orphan receptor gamma (γ) are expressed in CD4T cells rhythmically during dark phase and light phase, respectively (,). The development of regulatory T cells producing interleukin (IL)-10 is controlled by melatonin, suggesting that all coordination of the immune response is under circadian control (). ^{30 31 32 33 34 34} Nfil3 Ror t +

These results clearly show that the immune system is under the control of peripheral clocks. It is also regulated by hormones and neuro-mediators that reflect the activity of central pacemaker. The hypothalamic-pituitary-adrenal axis is activated in response to stress and appears synchronized to glucocorticoid circadian rhythms. Hence, oscillations of the lymphocyte number in humans are inversely correlated with diurnal rhythm of glucocorticoid production (). It is likely that the rhythmicity of the immune response involves numerous mechanisms, including the contribution of light variation. ³⁵

For a long time, it was believed that circadian rhythms are not expressed by single-cell organisms such as prokaryotes. One reason for this belief was the idea that organisms duplicating more than once a day do not need a rhythm longer than their life cycle (). In the 1980's, circadian rhythm was found present in cyanobacteria (–), photoautotrophic organisms that produce oxygen by photosynthesis as plants and eukaryotic algae (,). In cyanobacteria, photosynthesis occurs in daylight, whereas nitrogen production peaks during the night (). It has also been shown a circadian variation of the expression of most genes implicated in cell division, chromatin compaction and photosynthesis (). ^{36 37 39 40 41 42 43}

Mechanistic studies have enabled the identification of clock genes responsible for circadian rhythms have been recently discovered in most organisms including fungi, algae and photosynthetic bacteria; they are named, and, respectively (,). Regarding cyanobacteria, the central biochemical oscillator of their circadian rhythm is represented by three proteins encoded by thecluster (,,), SasA, CikA, and RepA, which control the timing training, and output of the cyanobacterial cell signaling process (). Several studies have examined how functions the circadian loop of cyanobacteria (,,–). A theoretical model of theoscillator suggests that ATP hydrolysis is a driving mechanism of phosphorylation oscillations and that the frequency of ATP hydrolysis in individualmolecules is correlated with the circadian rhythm frequency () (). The reconstitution of cyanobacterium circadian loopis associated with oscillations of Kai proteins (,), validating the theoretical approach of theoscillator. Frq, Ibp Kai KaiABC KaiABC KaiC in vitro KaiABC ^{44 45 10 46 47 48 40 42 49 51 50} Figure 2 ^{52 53}

The belief that circadian rhythms are specific of cyanobacteria and that eubacteria are “non-circadian” should be revisited. Using a bioinformatics approach, homologs ofandgenes have been found in non-circadian bacteria includingspecies and in archaea (). The functions of non-cyanobacterial Kai proteins have not been described, and it remains unknown whether these proteins interact with input and output factors (,). However, theoscillator, when expressed into the “non-circadian” bacterium, remains functional (), suggesting that “non-circadian” bacteria possess the armaturium needed to circadian rhythms. We recently identified thegene fromas astructural homolog using a bioinformatic approach and showed the persistence of circadian rhythm inandmutants, suggesting thatis not the generator of circadian rhythm of. Hence, it does not play the same role thanin cyanobacteria (unpublished data). To date, the identification of circadian rhythms in eubacteria remains challenging for the scientific community and opens new insights in terms of antibiotic resistance that could provide an interesting new therapeutic approach. KaiB KaiC Pseudomonas KaiABC Escherichia coli RadA E. coli KaiC RadA RecA RadA E. coli KaiC ^{46 54 55 56} −/− −/−

The circadian rhythms have been also described in parasites. (). With a blood stage lasting 24 h or multiple of 24 h, its rhythm was found to be associated with recurrent fever. It has been reported that circadian variations of parasites are influenced by variations of glycemia from high levels during the night to low levels during the day (,). Therefore, the links between rhythmicity of parasites and host homeostasis have been suggested. Similarly, the appearance of filarial parasites is also rhythmic (). The parasites have their own circadian rhythm, as exemplified bythat can generate circadian expression in the absence of host cells (). Plasmodium sp Trypanosoma brucei ^{57 58 59 60 61}

The insect vectors have their own biological rhythm () that depends on the response to daily light/dark cycle and molecular clock activity.mosquitoes that transmit malaria are active at dusk, so their bites occur at night during the host's resting phase. In contrast,mosquitoes that transmit dengue/yellow fever are day biters (). The pathogens may affect vector circadian rhythms. The dengue virus is responsible for high amplitude of rhythmic locomotor activity of mosquitoes.exhibits diurnal rhythms that contribute to circadian transmission of Zika virus (). The proteobacterium, an endosymbiont of arthropods, was reported to influence sleep time in flies (). Recently, it has been shown that the daily activity of fly is affected by peripheral clocks andpresence in flies (). ^{62 63 64 65 66} Anopheles Aedes Aedes aegypti Wolbachia Wolbachia

The existence of biological rhythms in vectors may affect their resistance to toxic compounds. In, the genes encoding the metabolic resistance to dichloro-diphenyl-trichloroethane (DDT) insecticide are rhythmically expressed (,). Correlations between circadian enzymatic detoxification of insecticide and feeding time have been established (). Thus, it appears that interactions between the circadian rhythms of the three partners involved in malaria transmission, namely mosquitoes, parasites and human host, lead to the development of the disease. Anopheles gambiae ^{67 68 69}

At the intersection of microbial world, immune response and homeostasis, the intestinal microbiota exhibit variations of circadian rhythms (,). The human gastrointestinal tract contains a large number of microorganisms, of which the most studied are the bacteria (). They contribute to maintain various host physiological functions such as immune defense and/or tolerance (). Recently, the circadian rhythm of the intestinal microbiome composition as well as the functions of this microbial community related to the circadian rhythm have been reviewed (). Although intestinal bacteria are not exposed to the light/dark cycle, they are subjected to circadian changes associated to host activities, including food intake and exposure to antibiotics (,). Nevertheless, light variations affect the abundance of some microbes such asknown to affect gut barrier integrity () and that ofsp. (). Interestingly, it has been reported that, a gram-negative bacterium, is sensitive to melatonin known to be a circadian rhythm synchronizer and expresses circadian swarming and motility (). These findings strengthen the hypothesis of circadian rhythms in prokaryotes. ^{70 71 72 73 74 74 75 76 77 78} Ruminococcus torques Clostridia Enterobacter aerogenes

The use of mouse models in which the intestinal microbiota is modulated or mice with a clock gene deficiency underlines the association between the microbiota and circadian rhythm. Liang et al. () found that the absolute number of fecal bacteria in C57BL/6 mice follows a circadian rhythm and that mice deficient ingene show an altered daytime rhythm of microbiota in a sex-dependent manner. Mice deficient inand disrupted sleep cycle show an almost total loss of rhythmic fluctuations of microbiota and present intestinal dysbiosis (). The circadian rhythms of microbiota over 24-h periods also depend on the host. Hence, this rhythmicity disappearswhen bacteria are cultivated, suggesting a regulation by the host (). Leone et al. () showed that the relationship between microbiota and circadian rhythms is affected by the type of diet in germ-free mice. Indeed, bacteria from the familyexhibit circadian rhythms under regular low-fat chow, which are absent in mice with high fat diet. The diabetic mice exhibit a loss of diurnal rhythms of bacteria such as(). ^{79 80 81 82 83} Bmal1 Per1/2 in vitro Lachnospiraceae Akkermansia genus

The association between circadian variations of microbiota and mechanisms of nutrition-associated disorders has been recently reported. Based on the observation that circadian misalignment promotes the occurrence of obesity, it has been shown that disruption of circadian rhythms impacts gut microbial composition and risk of obesity in rodents. In the absence of microbiota, the recruitment of histone deacetylase 3 is defective and cyclical histone acetylation is lost, suggesting a relationship between microbiota rhythms and epigenetic changes (,). In addition, a new target of diurnal rhythms of microbiota, the group 3 innate lymphoid cells (ILC3), has been recently reported (). Indeed, a dysregulation of brain rhythmicity affects circadian rhythms of ILC3 and microbiota, suggesting that environmental light directly regulates diurnal variations of enteric ILC3. In summary, a disruption of the circadian rhythm, by mutation of clock genes or disruption of the wake/sleep cycle, leads to changes in the composition of the intestinal microbiota and metabolic changes in host, opening a new fascinating field of investigations. ^{84 85 86}

The evidence of circadian variations in infectious diseases due to bacteria, parasites and virus is based on clinical observations and on the use of animal models including mice invalidated for clock genes (). Table 1

During infection, central and peripheral circadian rhythms of the host may be altered. In infected mice, the interaction ofwith epithelial cells account for circadian variations of pulmonary inflammation including the release of inflammatory mediators and recruitment of inflammatory cells. In the absence ofgene, the recruitment of neutrophils is increased in relation with a disruption of CXCL5-glucocorticoid receptor interaction (). Moreover, the infection with, that is responsible for gastritis and paves the way of gastric cancer, involves alterations in gastric acid secretion, which is regulated by circadian rhythm ().dysregulates the molecular clock in gastritis through the upregulation ofgene expression in gastric epithelial cells. Importantly, the expression of thegene is also upregulated in gastric tissues from patients with atrophic gastritis and dramatically increased in patients with precancerous lesions, thus establishing a relationship between disrupted circadian rhythm and the severity of the infection. In addition,disrupts the circadian rhythm of an importanttarget, the gene encoding tumor necrosis factor (), exacerbating the TNF production (). Streptococcus pneumonia Bmal1 Helicobacter pylori H. pylori Bmal1 Bmal1 H. pylori Bmal1 Tnf ^{87 89 90}

Disruption of circadian rhythm was also observed during intracellular bacterial infection.is a foodborne gram-positive bacillus known to be pathogen when T-cell mediated responses are impaired. The number of inflammatory monocytes considered as the effectors of inflammation and anti-immunity vary according to a circadian rhythm. Their circadian traffic to the tissues is disrupted by the myeloid impairment ofgene (). Our team previously showed expression variations of clock genes in Q fever, a zoonosis due to, an obligate intracellular bacterium. The analysis of microarrays performed to understand sexual dimorphism ininfection shows that circadian genes (, and) are upregulated in the liver from infected female mice, as compared with healthy females and infected males (). In patients, thegene is more expressed in males with acute Q fever than in healthy volunteers (), suggesting that the unexpected relationship between circadian rhythm and gender dimorphism of Q fever may be extended to other human infectious diseases. Listeria monocytogenes Listeria Bmal1 Coxiella burnetii C. burnetii Bmal1, Clock Per2 Per2 ^{91 100 101}

Interestingly, it was reported that parasites seem to be able to synchronize circadian rhythm and immune response. Indeed, in mice infected by, the frequency of parasite replication is related to the circadian rhythm of TNF expression (). Indeed, increased TNF is associated with a higher frequency of non-replicating cyclic forms of trophozoites. Infection alters circadian rhythms of blood glucose with a hypoglycemia at the end of the active phase and a direct impact on parasites. The oscillations of TNF and glucose levels seemed critical for parasite replication (). It is established that malaria due tosp. exhibit a periodicity of fever that is secondary to the burst of-infected red blood cells every 24–72 h (). Mice infected withhave a period shorter than 24 h and abnormal activity during the resting phase. This model reproduces sleeping sickness considered a circadian disorder in which infected individuals experience somnolence during the day and insomnia during the night (). Plasmodium chabaudi Plasmodium Plasmodium T. brucei ^{59 58 58 102}

Different viruses alter circadian-regulated biological processes such as CD4T cell numeration in human immunodeficiency virus infection (). Other interferences have been described for simian immunodeficiency virus, coxsackievirus A16 (hand, foot and mouth disease) and human T-lymphotropic virus (). Liver circadian-regulated genes that are likely targets for hepatitis C virus (HCV) may affect viral hepatitis. Patients with chronic HCV infection develop a disrupted circadian rhythm characterized by altered sleep patterns, although the association of sleep disorders and reduced survival in patients is debated (). HCV infection also leads to decreased expression ofandgenes. Thegene is critical for the anti-viral response since its overexpression in a hepatocyte cell line reduces HCV replication and increases the expression of interferon-stimulated genes (). On the other hand, liver-associated microRNA (miR)-122 is involved in HCV replication; miR122 is negatively regulated byα, strengthening the idea that HCV replication depends on the daily rhythm (). The association between hepatitis and circadian rhythms has potential implications for the patient management. Hence, viral RNA levels rebound more frequently in patients infected with HCV and undergoing liver transplantation in the morning than in those transplanted in the afternoon (). Hepatic circadian gene oscillation is associated with circadian rhythm and sleep in HCV infection (). + ^{103 96 94 95 104 105 94} Per2 Cry2 Per2 Rev-erb

Patients infected with respiratory viruses exhibit daily rhythms of clinical symptoms, such as increased nasal secretion and body temperature in the morning compared to the late evening in patients with cold or flu (). Mice with deficient molecular clock are highly susceptible to virus infection, as shown by increased replication of influenza, respiratory syncytial and parainfluenza type 3 viruses. Hence,appears as a major clock gene controlling viral replication (). Thegene is also involved in the coordination of lung immune response to virus since the deletion ofgene exacerbates acute bronchiolitis secondary tovirus orvirus (). In the same extend, the severity of influenza A infection is directly controlled by clock genes expressed in lung epithelium and natural killer (NK)1.1cells, a subset of NK cells (). The alteration of host circadian rhythm was also reported forvirus (HSV). The pathogenicity of HSV results from the interplay of viruses with circadian rhythms. The replication of HSV depends on histone deacetylation, and transcriptional machinery of HSV is associated with histone acetyltransferase, a clock-controlled gene (). ^{106 107 96 108 109} Bmal1 Bmal1 Bmal1 Sendai influenza A Herpes simplex +

Several studies reported that the susceptibility and the resistance to infections are associated with host circadian rhythm. More than 40 years ago, it was reported that susceptibility tois altered in blind and adrenalectomized mice with altered circadian rhythms (). Recently, Kitchen et al. () reported that macrophages frominfected-deficient-mice presented increase bacterial ingestion and cytoskeletal change both associated with an impaired function of RhoA pathway. The infection is more severe when mice are infected at the beginning of the resting phase than during the active phase (). In contrast, an increased number ofis found in mouse tissues at the onset of the resting phase as compared with the beginning of the active phase ().serovaris a foodborne microorganism whose recovery depends on the cell-mediated immune response. In mice, bacterial colonization of the colon is more pronounced during the resting phase than during the active phase. The inflammation induced bysp. varies similarly. This difference between resting and active time is abolished in clock mutant mice (). The circadian rhythm pattern ofinfection reflects circadian rhythm of the innate immune cells. Hence, the ingestion ofbacteria by peritoneal macrophages is maximum 16 h after serum-mediated synchronization (). S. pneumoniae S. pneumonia Bmal1 L. monocytogenes Salmonella enterica Typhimurium Salmonella S. typhimurium S. typhimurium ^{110 88 87 91 93 111}

In infection due to, it has been shown that cough, a major symptom of tuberculosis, is more frequent during daytime. This is related to highest sputum bacillary load (). In a mice model, rhythmic melatonin release generated circadian rhythms in granulomatous lesions after inoculation with BCG, an attenuated strain ofused as a vaccine against(). Moreover, it has been shown that the expression of metalloproteinases by peritoneal macrophages and spleen cells in response toinfection is controlled by the circadian clock in a-dependent manner (). Surprisingly, although it is well-established that corticosteroids increase susceptibility to mycobacteria, there is no consensus on the relationship between cortisol circadian rhythms and tuberculosis (,). Fever oscillations during daytime have been reported in Rocky Mountain spotted fever due to. They consist of an elevated temperature in the evening with a decline early in the morning, while the number ofDNA copies is elevated in the blood of patients collected early in the morning, suggesting that rickettsemia has a profile release similar to that of cortisol (). Mycobacterium tuberculosis M. bovis M. tuberculosis infection M. tuberculosis Bmal1 Rickettsia rickettsii R. rickettsii ^{92 112 113 114 115 116}

Murine models of parasite infection confirm the involvement of circadian rhythm in the evolution of several parasitic infections (). The moment of infection with the wormaffects the kinetics of worm expulsion. Hence, mice infected in the morning expel the parasite early, whereas the parasite persists in mice infected during the night. When thegene is deleted in antigen-presenting dendritic cells (DCs), the relationship of helminth expulsion and circadian rhythm disappears (). ^{98 99} Trichuris muris Bmal1

The relation between circadian rhythm and the regulation of the viral infection was also reported. Mouse models of infection exhibit daily variations in virus susceptibility: mice inoculated withvirus at the beginning of the resting phase (in the daytime) exhibit increased virus load as compared with mice inoculated during the active phase (in the nighttime) (). Recently it has been shown in a murine model that the severity of influenza An infection depends on the intrinsic pathogenicity of the virus and the uncontrolled inflammatory response. Mice infected before the onset of active phase exhibit higher mortality and morbidity than mice infected before the rest phase. When thegene is invalidated, the ability to fight the infection is lost through the hyper-inflammation induced by infection at the onset of the active phase (). Herpes Bmal1 ^{97 108}

Sepsis is an inflammatory response syndrome that appears after infection, or without documented infection (). It is the leading cause of death of patients in intensive care units () and there is some evidence that circadian rhythm and sepsis are tightly associated (). ^{117 118 119}

Murine models of endotoxemia that mimic human sepsis provide important information on the mechanisms involved in sepsis and the association between sepsis and circadian rhythm. The administration of lipopolysaccharide (LPS) to mice induces a systemic inflammatory response in which cytokine and chemokine productions are critical. The production of inflammatory molecules including IL-6, IL-12p40, and chemokines, such as CCL2, CCL5, and CXCL1 in response to LPS is higher in mice inoculated early in the active phase than in mice inoculated during the resting phase (). The endotoxemia in rats is associated with higher levels of inflammatory markers during nighttime than during daytime. In the presence of melatonin, daytime levels of inflammatory markers are increased, underlying the role of melatonin in circadian rhythm coordination in response to LPS (). ^{120 121}

The endotoxemia in young human volunteers is characterized by high levels of IL-10, an anti-inflammatory cytokine, during daytime and high levels of inflammatory cytokines (TNF, IL-1, and IL-6) and cytokine receptors during the nighttime (). This is consistent with placebo-controlled design studies that show that males receiving LPS in the evening exhibit higher rectal temperature and inflammatory mediator production than those receiving LPS in the morning when cortisol production is the highest (). There is evidence that LPS response depends on molecular oscillators. Hence, LPS injection induces a lesser production of cytokines inbone marrow-derived macrophages than in wild-type cells (). The engagement of Toll-like receptor (TLR)-9 induces circadian rhythm of TNF and CCL2 with a peak during the active phase; cytokine rhythms are disrupted when thegene is invalidated (). The genes of the molecular clock directly interact promoter regions of TLR genes, thus leading to their circadian rhythm (). ^{122 123 93 124 21} Clock Per2 −/−

Clinical studies suggest an association between circadian rhythm disruption and the development of sepsis. In a prospective study, our group assessed the circadian rhythm of cortisol and immune cells in 38 patients at day 2 after severe trauma. The trauma patients who develop an episode of sepsis later in their stay in the intensive care unit were compared to those who did not develop sepsis. The septic patients had higher levels of cortisol than the non-septic patients and delayed acrophases (ie., the peak of production during the period). The acrophases significantly differed between the two groups for lymphocytes, IL-10, and TNF (). This suggests that the primary insult, represented here by trauma, is associated with disruption of circadian rhythm that affects the host response. In addition, admission to intensive care units is associated with sleep disturbance, sedative infusions and loss of the daily light/dark cycle (). To counteract these adverse events, the use of melatonin was assessed in three randomized controlled trials (–). As the results of these three trials were disappointing, experts made no recommendation on melatonin use in intensive care units (). Taken together, these findings suggest that circadian disruption is not only associated to sleep dependent of the melatonin, but is multifactorial, and that the initial inflammatory process seems much more critical than the hospitalization in intensive care units. ^{125 126 127 129 130}