Planarian finds time(less) to fight infection

Planarians are freshwater non-parasitic flatworms that have fascinated researchers for more than 200 years.¹ Planarians have incredible capacities for whole body regeneration. While humans or mice have only limited potential to regenerate their tissues (mainly skin, liver and bone marrow), the planarian flatworm is able to regenerate a complete organism including its surprisingly complex brain from a tiny piece of its body within 7–10 days.^2-5 This is due to the fact that different to humans or mice who have a very limited number of stem cells distributed in stem cell niches in very specific regions of the body, planarians possess many adult somatic stem cells called neoblasts distributed along the whole planarian body. Neoblasts account for approximately 25% of the total number of cells in the parenchyma⁶ and include a population of pluripotent stem cells.⁷ Often planarians present homologs for human genes that are not present either in Drosophila melanogaster or Caenorhabditis elegans. Technological advances to aid in the study of planarians include genomics, transcriptomics (including single-cell transcriptomics), proteomics, high-throughput RNAi-screens, behavioral platforms (learning and memory), and drug toxicity assays.^8-20 Altogether the planarian model has become an excellent system for investigating in vivo stem cell biology and regeneration and it is an emerging biomedical model to study cancer and diverse human pathologies.^21-25

Although the immune system has been described as an important modulator of wound repair and regeneration^26-28 it is largely unexplored in planarians. It is likely that the mucus that covers the planarian body and whose production is greatly increased after wounding has anti-microbial properties.^29,30 Proteomic analyses have shown that planarian mucus proteins display similarities with the ones in human mucosal secretions³¹ and that the introduction of bacterial endotoxins into planarian wounds induces several potential immunity related genes, some of which share sequence similarity with vertebrate innate immunity genes.³² Phagocytic activity has also been observed in planarians by means of electron microscopy where putative phagocytic cells called reticular cells have been observed phagocytosing bacteria and also cellular debris during regeneration.^33,34 Finally, it has been discovered that the planarian genome contains many potential homologs of genes related to innate immunity and that some of them are activated during regeneration.^35,36 Recently two publications have shown the potential of the planarian model to become an excellent system for investigating immunity. The first one comes also from the laboratory of Eric Ghigo³⁷ in which they demonstrate that planarians are highly resistant to infection with bacteria pathogenic to Homo sapiens, C. elegans and/or D. melanogaster and thus are a good model to identify novel innate resistance mechanisms in humans. Indeed, after performing whole-transcriptome analysis coupled with an RNAi screen in planarians challenged with bacteria, they were able to identify the novel regulator of mammalian LC3-associated phagocytosis MORN2. The second study elucidated the composition of the planarian microbiome.³⁸ They observed that culture conditions that increase susceptibility to tissue lesions or the process of wounding itself coincided with an increase in Proteobacteria. Interestingly, they found that the elucidated planarian microbiome has a distribution of bacterial phyla similar to the human lower intestinal microbiome. This phyla composition is not present in other invertebrate or vertebrate systems that are consolidated models for the study of immunity and host-microbe interactions.^39-41

In this issue of Virulence, the laboratory of Eric Ghigo (Tsoumtsa et al.)⁴² set out to investigate how the circadian clock regulates the capacity of planarians to fight infection. The circadian clock is an internal time-keeping regulator present in most if not all of the cells of most organisms. It regulates many physiological processes in eukaryotes, from sleep/wake cycles, metabolism, body temperature, blood pressure or cardiovascular function in mammals to growth and photosynthesis in plants. The photoperiod is the most important Zeitgeber (time giver) for circadian oscillators in all investigated organisms so far. In mammals the central oscillator is the suprachiasmatic nucleus (SCN) located at the base of the hypothalamus in the brain, which is in charge of coordinating more peripheral oscillators present in the cells of many other organs. The circadian rhythm is maintained through 24 hours oscillating transcriptional-translational feedback loops that regulate the rhythmic expression of thousands of genes, which is translated into rhythms in metabolism and behavior.^43,44 Currently it is known that RNA-level post-transcription mechanisms, such as mRNA polyadenylation⁴⁵ and RNA methylation,⁴⁶ are at least as important regulators of the circadian clock as the activation/repression of transcription. The vertebrate primary feedback loop includes among others the transcription factors BMAL1 (also called in vertebrates ARNTL1 or Cycle in Drosophila), CLOCK, PERIOD (PER1, PER2 and PER3) and two CRYPTOCHROME proteins (CRY1 and CRY2).^47,48 These components are not always all present in other peripheral oscillators of other tissues or other organisms or even if expressed, other transcription factors may play their role. For instance, in Drosophila the function of CRY is performed by Timeless (Tim),⁴⁹ although in mammals there is a TIMELESS-LIKE protein, which has other functions in addition to regulating the circadian clock. In the same way, in the forebrain of mice, NPAS acts as a functional substitute for CLOCK.⁴³

Although the circadian rhythm may be an important regulator of diverse physiological functions in planarians, it remains largely unstudied. The evidences for a planarian circadian rhythm are few. One evidence is the presence of a 24-hour cycle of melatonin production. In vertebrates, one of the main functions of melatonin is as a hormonal output of the circadian clock in the SCN in the brain. Circadian clock genes in the SCN control melatonin production through signaling to the pineal gland, which secretes melatonin that signals in photoperiod to the other peripheral clocks in the body.⁴⁷ It has been suggested that planarians possess a circadian clock controlling melatonin synthesis, since melatonin and both its precursor serotonin and its synthesizing enzymes N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) fluctuate following a diurnal variation, peaking during the dark period.^50-52 In addition, it has been shown that planarian asexual reproduction through fission follows photoperiodicity in a day-night rhythm with reproduction occurring preferentially at night. Furthermore, fissioning is inhibited when maintaining planarians under continuous light.^53,54 It has also been hypothesized that melatonin release from the brain may mediate the influence of environmental photoperiods on fissioning since amputation of the planarian head breaks this rhythm and increases the fissioning rate⁵⁰

Tsoumtsa et al. seek for circadian genes in the planarian genome that regulate immunity by searching for those whose downregulation was previously linked in other model systems to an increase in the susceptibility to bacteria infection. They found homologs for Arntl1 (Smed-arntl-1 in planarians) and for Timeless (Smed-tim in planarians) but they could not find either Clock or Per-2. Thus, at least Clock may have been lost in the planarian lineage, since a Clock gene has been reported in the cnidarian Nematostella vectensis.⁵⁵ A requirement for those genes being part of the circadian clock is that their expression shows a 24 hours expression cycle. Indeed, they observed that Smed-tim expression changes during light/dark (L/D) conditions with a maximum peak of expression in the dark phase in a similar way to what happens in Drosophila.⁵⁶ On the other hand, when planarians were entrained to a L/D condition and then exposed to a dark/dark (D/D) condition they showed a desynchronized Smed-tim expression. Although clear daily oscillations in melatonin production are present in many protostomes including planarians, a clear association between circadian clock genes and melatonin production has been done only in vertebrates and recently also in the cnidarian Nematostella vectensis.⁵⁷ A possible experiment in planarians that would clarify this relationship would consist in trying to reset the circadian pattern of expression of Smed-tim in D/D conditions by exposing animals to melatonin, as reported in Nematostella.⁵⁷ Altogether these are very promising results that further support the idea of a circadian clock in planarians. It would be interesting to obtain the transcriptional profile of planarians at different time points during the L/D cycle. This would help to identify which physiological processes in addition to asexual reproduction, the circadian clock is regulating in planarians and to better understand the workings of the planarian circadian clock.

The main focus of Tsoumtsa et al. is to find out the relationship between the circadian clock genes and infection. Emerging evidence indicates that the immune system is under tight regulation of the circadian clock. It has been observed that perturbations of circadian rhythms impair immune function and thus health. For instance, epidemiological studies on shift-workers have shown that working during the night increases the risk for several cancers, obesity, diabetes, and cardiovascular problems. It is known that neural and endocrine signals transmit circadian information to immune tissues and that immune cells express molecular clock components to mediate immune responses including NK cytotoxicity, phagocytosis, and inflamation.⁵⁸ In vertebrates the use of clock genes mutant mice exposed to bacterial infection has demonstrated their role in immune function. It is known that circadian clock genes regulate the expression of several transcription factors involved in the immune system, including Stat3 and Stat5, Egr1 and NF-kB, and also control NK cell and macrophage activities.^44,58 In invertebrates there are also many evidences showing that circadian clock genes control the immune system. These are based on Drosophila mutants for clock genes, which have been or not infected with bacteria. For instance, in Drosophila the resistance to Staphylococcus pneumoniae oscillates daily. However, these oscillations are absent in Tim mutants. Indeed, it was shown that Tim regulates phagocytosis, with a maximum activity at night. While Tim mutants have normal phagocytic activity during the day, they lack the night peak.⁵⁶ Tsoumtsa et al. followed a similar approach to the one described here. The authors kept planarians under a L/D cycle or under a D/D cycle and then downregulated Smed-tim by RNA interference (RNAi). Planarians were then fed with Staphylococcus aureus. The authors could observe that Smed-tim RNAi treated planarians took longer than controls to eliminate S. aureus when kept under L/D conditions. Under D/D conditions the time to clear S. aureus was similar to controls. They concluded that Smed-tim is necessary for efficiently eliminate S. aureus under L/D conditions (Fig. 1). The authors also sought into the mechanism that Smed-tim signals to clear S. aureus. They found that the expression of antimicrobial genes p38 map-kinase, traf6 and morn2 in planarians are significantly induced after S. aureus infection. However, Smed-tim RNAi planarians had a significant decrease only in the expression of morn2 and traf6 and only under L/D conditions. Open questions remains as to if Smed-tim regulates resistance to other bacteria and whether Smed-arntl-1 has any antibacterial activity even though it does not regulate resistance to S. aureus. It would also be interesting to know whether phagocytosis is the main process regulated by Smed-tim and whether a peak of phagocytosis exists during the night.

Figure 1.

The antimicrobial response of planarians against Staphylococcus aureus is under the control of timeless. During light/dark cycles, planarians are able to eliminate S. aureus in 6 days through the induction of Smed-tim. Smed-tim promotes the antibacterial response by regulating Smed-traf6 and Smed-morn2.

The study of Tsoumtsa et al. uncovers a function of the gene timeless as a member of the planarian circadian clock and regulator of immune response to S. aureus in planarians. The paper opens up for further work not only into the relationship between the circadian clock and immunity but also into the functions and mechanisms of the circadian clock in planarians. It also contributes in consolidating the planarian system as a model for the study of immunity.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.