**3. Review Methodology**

We conducted a literature search on PubMed by using the following keywords: (1) *Caenorhabditis elegans* and probiotics; (2) *Caenorhabditis elegans* and lactobacilli; (3) *Caenorhabditis elegans* and bifidobacteria; and (4) *Caenorhabditis elegans* and lactic acid bacteria. Publication dates were restricted to the last ten years. The first search retrieved 46 results. The other searches produced a majority of overlapping results with the first one and some additional results, in particular: the second search retrieved six more results; the third one two more results and the fourth one three more results. Following this initial search, eight articles were excluded, on the basis of their low adherence to the description of probiotic activities in *C. elegans*, which was the main focus of our search. Thus, finally 49 articles were carefully evaluated for the review preparation. Table 1 shows a list of the microorganism strains described in the selected literature to exert a probiotic activity in *C. elegans*. The majority of the tested species belonged to the *Lactobacillus* genus, with 16 different species and 35 strains, followed by the *Bifidobacterium* genus, with 4 species and 6 strains. Then we found 2 *Pediococcus*, 2 *Weissella* and 2 *Enterococcus* species, and finally *Bacillus, Butyricicoccus, Megasphaera, Clostridium, Propionibacterium, Escherichia* and *Kluyveromyces*, with one species each. Of note, only *Kluyveromyces marxianus* belongs to the Ascomycota phylum within the yeast kingdom.

Among the collected papers, those taking into account the effect of probiotic supplementation on nematode lifespan were further selected and analyzed to evaluate important parameters related to ageing, such as oxidative stress, immune system and susceptibility to pathogen infection, which are known to be involved in immunosenescence. This condition refers to the gradual deterioration of the immune system brought on by age progression. It involves both the host capacity to respond to infections and the development of long-term immunological memory. Immunosenescence can be considered as a crucial contributory factor to the increased occurrence of morbidity and mortality among the elderly. This age-related immune deficiency is ubiquitous and found in both long- and short-living species, and it is characterized by a particular "remodeling" of the immune system, in duced by oxidative stress [27]. Together with inflammageing, immunosenescence is suggested to stand at the origin of the majority of elderly-related alterations, such as infections, cancer, autoimmune disorders, and chronic inflammatory diseases [28]. The present review focuses on the role of probiotics on *C. elegans* age-related parameters, with particular emphasis on oxidative stress, immunity and inflammation, and protection from pathogen infections.


**Table 1.** List of microbial strains reported in the selected literature to exert a probiotic activity in *C. elegans*.


**Table 1.** *Cont.*

<sup>1</sup> The current adscription is *Bifidobacterium longum* subsp. *infantis* [70].

#### **4. Mechanisms Involved in** *C. elegans* **Lifespan Extension Induced by Probiotics**

#### *4.1. Description of the Main Pathways*

The principal pathways involved in lifespan control, oxidative stress, regulation of immune response and defense against pathogen infection in *C. elegans* include the IIS pathway and p38 MAPK pathway [71]. Each pathway is composed of a cascade of signaling molecules that finally activate/regulate the transcription of specific target genes. In particular, the IIS pathway is initiated by the activation of dauer formation (DAF)-2, an insulin/insulin-like growth factor-1 receptor ortholog, subsequently triggering a cascade of phosphorylation events that activate specific kinases and downstream mediators. These include phosphatidylinositol 3-kinase AGE-1, phosphoinositide-dependent kinase (PDK)-1, and various serine/threonine protein kinases (AKT-1, AKT-2, and SGK-1), culminating in phosphorylation of DAF-16, a protein belonging to class O of the forkhead transcription factors (FOXO), resulting in its inactivation [72]. On the contrary, in the presence of heat stress, anoxia, oxidative stress, starvation, and infections, the IIS pathway is down-regulated and DAF-16 migrates to the nucleus, where it switches on the expression of specific target genes, that contribute to several cellular processes, from apoptosis to stress resistance, prolongevity and anti-ageing [58,73]. The nuclear translocation of DAF-16 leads to both up-regulation and down-regulation of large sets of genes, referred to as class I and II, respectively [5]. The IIS signaling pathway transcriptionally regulates many genes involved also in the immune responses, closely linked to longevity in *C. elegans*.

The p38 MAPK pathway is the most ancient signal transduction cascade in nematode immunity and plays a central role in *C. elegans*response against different pathogens, as it does in mammals. The p38 MAPK pathway is required for the activation of a set of immune effectors necessary to maintain a basal level of immune function and it is also involved in lifespan extension [67,74]. A neuronal symmetry (NSY)-1–SAPK/ERK kinase (SEK-1)–p38 mitogen-activated protein kinase ortholog (PMK)-1 p38 MAPK cascade (MAPKKK-MAPKK-MAPK, respectively) was elegantly identified as a key component of the *C. elegans* immune response [6].

Such signaling pathways are evolutionarily conserved in different animal species, from nematodes and flies to higher vertebrates and mammals. Evidence suggests that these pathways are relevant also to mammalian aging [75]. In particular, human studies conducted on centenarians highlighted an important role of the IIS pathway in setting lifespan, since associations have been found between polymorphisms in IIS genes and longevity [76].

Probiotic strains used in *C. elegans* studies have been shown to act through one or more of the above mentioned signaling pathways (Figure 2 and Table 1).

**Figure 2.** Schematic representation of the most common signaling pathways influenced by probiotic strains employed in *C. elegans* studies. Each pathway is represented by a distinct color gradient. The list of single bacterial strains influencing the different pathways is reported in Table 1. Abbreviations used: AMPK: 5 AMP-activated protein kinase; AKT-1/2: serine/threonine protein kinase orthologs; BAR-1: beta-catenin/armadillo Related-1; DAF: dauer formation; DBL-1: DPP/BMP-Like-1; HSF-1: heat-shock transcription factor-1; IIS: insulin/insulin-like growth factor-1; JKK-1: c-Jun N-terminal kinase kinase; JNK-1: c-Jun N-terminal kinase; MAPK: mitogen-activated protein kinase; NSY-1: neuronal symmetry-1; PDK-1: phosphoinositide-dependent kinase 1; PMK-1: p38 mitogen-activated protein kinase-1 ortholog; RACK-1: receptor activated protein C kinase; SEK-1: SAPK/ERK kinase-1; SGK-1: serine/threonine protein kinase ortholog; SKN-1: skinhead family member-1; SMA: small; TIR-1: toll interleukin-1 receptor-1.

## *4.2. Oxidative Stress Response*

Oxidative stress plays a detrimental role in different organisms. Normally, antioxidant defenses protect cells by removing reactive oxygen species (ROS). During ageing, on the other hand, ROS and other products of oxygen metabolism accumulate damaging proteins, lipids and DNA, and weaken antioxidant defenses [77]. In*C. elegans* animal model, several studies have been carried out to understand the mechanisms through which probiotics can influence ageing, by activating different longevity signaling pathways related to oxidative stress resistance. Prolongevity and oxidative stress responses in *C. elegans* fed probiotics are induced via mechanism(s) that can be DAF-2/DAF-16-dependent or a result of a cross talk among different pathways.

Among the tested strains, *Bifidobacterium longum* strain BR-108 has been shown to increase worm lifespan following H2O2-induced oxidative stress, through activation of IIS pathway. After the cascade activation, DAF-16 seems to co-localize with the heat-shock transcription factor (HSF)-1 in the nucleus, in ducing the transcription of *hsp-16.2* and *hsp-70* that are involved in stress responses and longevity [59].

Similarly, *Lactobacillus rhamnosus* CNCM I-3690 and *Bifidobacterium animalis* subsp. *lactis* CECT8145 strains stimulated a strong resistance to oxidative stress in *C. elegans*, which was in part dependent on the IIS pathway [49,53]. On the other hand, *Lactobacillus gasseri* SBT2055 (LG2055) has been reported to promote a prolongevity effect in *daf-2* and *daf-16* mutant worms [9], thus demonstrating that prolongevity and enhancement of stress resistance were DAF-16-independent. These phenotypes occur rather by triggering the p38 MAPK pathway, which culminates with the nuclear translocation of the transcriptional factor skinhead family member (SKN)-1 [9]. SKN-1, an ortholog of the mammalian Nrf2, in duces the expression of target genes involved in oxidative stress resistance and it is responsible for the beneficial effect exerted by several other probiotic microbes [37,62,69].

*Bifidobacterium longum subsp. infantis* (formerly *B. infantis*) strain ATCC15697 resulted to extend wild-type nematode lifespan, but it failed to prolong the lifespan of *pmk-1, skn-1* and also *daf-2* mutants, demonstrating the involvement of p38 MAPK and IIS signaling pathways, both modulating SKN-1 activation [56,78].

It has been reported that the c-Jun N-terminal kinase (JNK) family, a subgroup of the MAPK superfamily, phosphorylates DAF-16 at a different site with respect to DAF-2-mediated phosphorylation, resulting in its nuclear translocation [79].

Similarly, AAK-2, which is one of the two alpha-catalytic subunits of 5 -AMP-activated protein kinase (AMPK), can directly phosphorylate DAF-16, triggering prolongevity and oxidative stress responses. Analysis of lifespan and gene expression of worms fed *Weissella koreensis* or *W. cibaria*, demonstrates that some *Weissella* species promote longevity in *C. elegans* by inducing oxidative stress responses through activation of DAF-16 via the JNK and AMPK pathways [68].

Several detoxifying enzymes are induced by different transcription factors in response to oxidative stress. Two of these are the superoxide dismutase (SOD) and the glutathione S-transferase (GST), which detoxify ROS [80]. Moreover, oxidative stress causes the activation of the transcriptional factor HSF-1, which also regulates lifespan, and activation of JNK pathway. Zhao and coworkers demonstrated that *Bifidobacterium longum* BB68, isolated from a centenarian subject, was able to increase lifespan and oxidative responses in *C. elegans,* through increased expression of *sod-3* gene, mediated by the toll interleukin-1 receptor (TIR)–JNK signal transduction pathway resulting in DAF-16 nuclear translocation [58]. Specifically, this highly conserved pathway consists of a TIR-domain protein, TIR-1, activating JNK-1 through phosphorylation. In turn, JNK-1 phosphorylates DAF-16, which migrates to the nucleus.

As stated above, in tracellular ROS represent an important marker to analyze the extent of oxidative stress, and some probiotic strains have been shown to reduce their level in *C. elegans*. *L. fermentum* MBC2, in addition to lifespan extension and anti-ageing effects, in duced a reduction of ROS levels and an increased expression of detoxifying enzymes, such as GST-4, paralleled by an amelioration of the other ageing biomarkers, such as locomotion activity, pumping rate and lipofuscin granules [36].

#### *4.3. Immune Response and Pathogen Protection*

Several candidate probiotic bacteria analyzed in this review have also been demonstrated to affect immunity and inflammation pathways in *C. elegans*. Immune response and lifespan are tightly linked in *C. elegans*. Among different molecular pathways shared with higher organisms, in nate immunity of *C. elegans* shows many aspects similar to humans. Although the nematode does not have a cell-mediated immune system, it possesses innate immune defense mechanisms that are evolutionarily conserved [74]. In particular, *C. elegans* possesses different pathways associated with immunity, in cluding the above mentioned p38 MAPK and IIS pathways, but also the trasforming growth factor-beta (TGF-beta) [52,71,74,81] and the beta-catenin signaling pathways [82], which can be induced by probiotics (Figure 2).

Immune responses to bacteria are mediated by interaction of specific microbial cell wall structures, (microbial associated molecular patterns, MAMPs), such as peptidoglycan, teichoic acids and lipopolysaccharides, with host receptors, in particular toll-like receptors (TLRs). In mammals different TLRs have selective specificity for the different MAMPs, while in *C. elegans* a unique TLRs homolog, TOL-1, has been identified so far [83]. The interaction of a TLR with its microbial ligand activates several signaling pathways, in cluding p38 MAPK, resulting in the transcription of genes necessary to mount the defense mechanism in the host. The main cell wall MAMPs share a common

basic structure among different bacterial species, both pathogens and probiotics, but various subtle chemical modifications present in the different species or strains can contribute to the strain-specific properties of probiotics. This also implies that the final outcome of the TLR activation depends on the type of interacting microorganism, meaning that a MAMP from one bacterial species can activate a certain TLR, while a similar MAMP from another species, or strain, can down-regulate the same TLR signaling [84]. The involvement of nematode TOL-1 in the regulation of prolongevity effect exerted by *Bifidobacterium longum* subsp. *infantis* (formerly *B. infantis*) strain ATCC15697 was recently demonstrated [57].

The majority of probiotic strains were employed in *C. elegans* to verify their prolongevity effects in the context of protection from pathogen infection, through killing assays. Some others were tested on lifespan extension in normal conditions. The use of nematode functional mutants or the analysis of gene expression profile by RT-qPCR/microarray allowed the elucidation of the molecular players acting as targets of probiotic action. Many human pathogens, such as *Pseudomonas aeruginosa*, *Salmonella enterica*, *Staphylococcus aureus*, *Klebsiella pneumoniae*, enterotoxigenic *Escherichia coli, Yersinia enterocolitica* and *Listeria monocytogenes*, can cause nematode death. It is known that pathogen infection induces worm innate immune responses, consisting in the production of several antimicrobial proteins, whose expressions are regulated by signaling pathways involved in the defense against harmful bacteria [74]. Such antimicrobial proteins include lysozyme (LYS) family, and C-type lectins (CLEC). As mentioned above, *C. elegans* lacks a cell-mediated immune system and the production of antimicrobial peptides is, therefore, the outcome of its innate immunity to counteract infections [85]. As an example, *Bacillus subtilis* NCIB3610, which forms a biofilm contributing to nematode prolongevity, specifically stimulated *lys-2* expression, in creasing *C. elegans* resistance to *P. aeruginosa* infection [86].

Zhou and coworkers reported that *L. reuteri* CL9 induced the expression of antimicrobial peptide genes *clec-60* and *clec-85*, in volved in the protection of nematodes against enterotoxigenic *Escherichia coli* (ETEC) infection [51]. Similarly, *L. zeae* LB1 induced the production of antimicrobial peptides and defensive molecules, such as LYS-7 and CLEC-85, through the p38 MAPK and IIS pathways, enhancing resistance of *C. elegans* to ETEC infection [52]. The p38 MAPK pathway was also activated by *L. acidophilus* NCFM, employed for protecting nematodes against the Gram-positive pathogens *Staphylococcus aureus* and *Enterococcus faecalis* [29], as well as by *L. casei* LAB9, which displayed protection against *Klebsiella pneumoniae* infection. In particular *L. casei* LAB9 activates TLR and triggers the PMK-1/p38 MAPK pathway through the up-regulation of receptor activated protein C kinase (RACK)-1, an adaptor molecule that plays a critical role in the host defence and survival [32].

The p38 MAPK pathway is also involved in protection against *Legionella pneumophila* infection promoted by *Bifidobacterium longum* subsp. *infantis* (formerly *B. infantis*) ATCC 15697 via PMK-1 [55], as well as in stimulation of *C. elegans* host defense by six foodborne strains of *Bacillus licheniformis* [87]. Moreover, Kwon et al. (2016) described that *Propionibacterium freudenreichii* KCTC 1063, isolated from a dairy product, in creased resistance against *Salmonella* typhimurium, through the activation of SKN-1, upon phosphorylation by PMK-1 [67].

*L. acidophilus* NCFM immune stimulation involved also the beta-catenin pathway through the beta-catenin/armadillo related (BAR)-1 mediator, in dicating that different signaling pathways can act in parallel to promote immunity [29].

On the other hand, the IIS signaling pathway was influenced by *Clostridium butyricum* MIYAIRI 588 (CBM 588), which was able to confer resistance to *S. aureus* and *S. enteric* infection through DAF-16-dependent class II genes [62]. Two other genes implicated in the defense response and the innate immune response through the IIS pathway, *acdh-1* and *cnc-2*, were up-regulated by heat-killed *L. plantarum* LP133 and *L. fermentum* LF21, protecting worms against Gram-negative pathogens *Salmonella* typhimurium and *Yersinia enterocolitica* [39].

The evidence that the protective activity of different *Lactobacillus* species can be directed either to Gram-positive or Gram-negative bacteria indicates that probiotic effects are species- and strain-specific, as explained above, concerning TLR-MAMP interactions. To this respect, the above-mentioned

*Lactobacillus acidophilus* strain NCFM, while active against Gram-positive bacteria, displayed a minimal inhibitory effect on Gram-negative infection with *P. aeruginosa* or *S. enterica* [29].

In the absence of pathogen infection, lifespan extension exerted by *L. salivarius* DSM 20555 resulted to be dependent on the up-regulation of *lys-7* and *thn-2* genes, encoding LYS and an immune effector member of the thaumatin family, respectively, in a DAF-16-independent manner, suggesting the involvement of pathways other than IIS signaling [47]. In line with this evidence, *Butyricicoccus pullicaecorum* KCTC 15070 and *Megasphaera elsdenii* KCTC 5187 prolonged *C. elegans* lifespan by activating nuclear receptor signaling and the innate immune system in a TGF-beta pathway-dependent, but IIS pathway-independent manner. The signaling involves DPP/BMP-Like (DBL)-1, a mediator of the TGF-beta pathway, which binds to TGF-beta receptors, such as SMA-6/DAF-4, on the cell membrane and activates transcription of specific target genes related to antibacterial defense, in ducing the production of antimicrobial peptides, such as CLEC, LYS and lipase [61].
