Leishmania, inbred strains of mice, SOCS and DNA hypermethylation

While most strains of inbred mice are able to control and largely eliminate parasitic L. major, BALB/c and DBA/2 mice develop non-healing lesions [1]. It has been clearly established that Th1-type cytokines such as IFN‑γ offer protection to the intracellular parasite, while Th2-type cytokines such as IL‑4 are linked to susceptibility [2-5]. Macrophages play a dual role in Leishmania infection because they are part of the innate immune system but also are the primary host cells for the parasite. It has been demonstrated that IFN‑γ drives macrophages to acquire the classically activated phenotype characterised by iNOS expression in murine cells, whereas IL‑4 stimulation of macrophages is recognised to drive them to an alternative state of activation typically expressing the enzyme arginase1 [6-8]. The activity of arginase1 has been correlated to parasite proliferation [9;10]. Arginase breaks down arginine to produce urea and ornithine, which then can be metabolised further to proline (a possible source of energy and a building block for protein synthesis in parasites), polyamines (DNA packaging) and possibly nucleotide biosynthesis (ornithine to glutamine to purine and/or ornithine via ornithine transcarbamoylase to pyrimidine bases?), all of which could or do act as nutrients for the proliferating parasite. (see: "http://www.nature.com/nri/journal/v2/n7/full/nri843.html")

In mice, iNOS plays a very important role in controlling the disease (humans have evolved other mechanisms of Th1 mediation, e.g. Vitamin D receptor-mediated mechanisms [11], which seem to be easily available to sun-exposed humans compared to nocturnal mice). Normally resistant mice that have been made deficient in iNOS become susceptible to L. major, while phox-deficient mice are able to control the disease, implying different modes of action by reactive oxygen species (ROS) generated by phox and reactive nitrogen intermediates (RNI) such as NO [12]. iNOS competes with arginase for the same substrate, arginine. But unlike arginase, iNOS produces citrulline and the radical NO which is not further metabolised to produce a source of energy, building blocks or nucleotide synthesis. In fact, a complex inter-regulatory network of competition and inhibition exists between arginase and iNOS on an enzymatic level (iNOS produces NOHA in the first step to NO catalysis, which acts as an inhibitor to arginase1 [13]) as well as on the complex cross-regulation of cytokines that induce these enzymes (reviewed elsewhere and in [14-16]).

The balance between these two enzymes is therefore of crucial importance to the parasite, with the activity leading to proliferation and the other to parasite killing.

I propose that the observed differences between the healing and non-healing strains of mice is due to different levels of suppression of the arginase-inducing cytokine signalling. In particular, I hypothesise that differences in SOCS expression, possibly due to DNA hypermethylation, are central elements controlling the outcome of the disease. The role of S-adenosylmethionine in this model system may offer clues to possible drug candidates not necessarily limited to parasitic diseases.

SOCS

SOCS proteins are a relatively recently discovered group of at least eight intracellular proteins that act as negative regulators of cell signalling [17-19]. Their SH2 domain binds to phosphorylated Tyrosine (pY) residues and disrupts cell signalling involving Tyrosine kinases, while their SOCS box-domain has been shown to have ubiquitin E3 ligase activity [20-24]. One major route for poly-ubiquitinated cytosolic proteins is their degradation by the proteasome while mono- or multi-ubiquitination of membrane proteins leads to the internalisation or even lysosomal degradation of these proteins [25]. (see: "http://www.nature.com/nri/journal/v2/n6/full/nri818.html")

Many major cytokine receptors such as IFN‑γR, and IL‑4R do not have an inherent Tyrosine kinase activity themselves, but ligand-induced dimerisation leads to recruitment and activation of Tyrosine kinases of the JAK family which in turn lead to the phosphorylation and activation of STAT transcription factors [19]. Tyrosine phosphorylation is a widely used messaging system for short-lived, reversible and regulated signal transduction. In addition to cytokines such as IFN‑γ and IL‑4, many other receptors use phosphorylation of Tyrosine residues as a signal. They either act directly as Tyrosine kinases themselves, (the insulin receptor phosphorylating itself and its substrates IRS1 and IRS2 see review by Youngren accepted for publication in CMLS 2007), or recruit Tyrosine kinases to phosphorylate downstream signalling adapters for them. Phosphorylation of proteins can also be used to facilitate the recruitment of individual components into larger signalling complexes held together and assembled in the correct order by pY – SH2 domain binding (for instance, it may be possible that phosphorylation of the TLR leads to its recruitment into complexes involving other signalling molecules such as PI3 kinases, adenylate cyclases or G-proteins or other proteins with kinase or lipase activity, not to mention other substrates [26;27]).

Members of the SOCS family (CIS and SOCS1 to SOCS7) have distinct but possibly also overlapping functions due to differences in expression (quantity, time, location) and affinity to their respective different target proteins not all of which have been characterised as yet. Many cytokine signals induce SOCS expression resulting in negative feedback either limiting the intensity/duration of the initial signal (IFN‑γ inducing SOCS1, which disrupts IFN‑γ signalling via STAT1) or rendering cells unresponsive to following stimuli (IFN‑γ-induced SOCS1 blocking IL‑4 signalling via STAT6) [19;28]. These proteins can confer protective as well as destructive effects on cell and tissue function/physiology: over-expression of SOCS1 in pancreatic cells can protect β-islet cells from IFN‑α/β-induced cell death (possible protection from type-I diabetes) [29], but over-expression of SOCS1 in muscle tissue can render the cells insulin-resistant (possibly leading to type-II diabetes) [30].

Generating knockout animals with disrupted SOCS genes has proven difficult in some cases, due to embryonic lethality for SOCS1 and SOCS3 expression. In the case of SOCS1, it was possible to obtain mice with SOCS1-deficient animals if they also were IFN‑γ-deficient [31-33]. SOCS3-deficient mice died because of placental defects [34].

Because they are induced by cytokines such as IL‑4 and IFN‑γ, it should come as no surprise that SOCS proteins are differentially expressed in different types of T helper cells, as indeed they are, playing an important regulatory role in the development of different cell types [35].

Toll-like receptors have also been shown to up-regulate SOCS proteins either directly through cell signalling involving TIR domain-containing adapter protein or indirectly through auto- and paracrine actions of induced cytokine production (such as type I interferon, IL‑6 and TNF‑α) [36] while endotoxin tolerance was not observed in SOCS1-deficient mice [37].

Recently, it had been shown that certain forms of cancer had switched the expression of some or many SOCS genes off [38-47]. The absence of the negative feedback mechanism allowed them to receive continuous and uninhibited growth signals via cytokine receptors such as IL‑6 (e.g. prostate cancer). Some breast cancer cell lines expressed high levels of a number of SOCS, but these were later shown to have a mutation in a STAT, which meant that the cells continued to receive growth signals in the absence of any external stimulus and despite the presence of SOCS proteins [41]. In fact it might be speculated that shutting down other cytokine signalling pathways ensured that the cells remained in an incompletely differentiated state and contributed to the malignancy. Where cancer cells had down-regulated the transcription of SOCS genes, it was often found that this had been achieved by hypermethylation of the relevant promoter sequence.

Mice

To my knowledge, no one has looked specifically for differences in SOCS expression in mice with the aim of comparing differences between strains of inbred mice. However, the following observations seem to suggest an involvement of SOCS:

1) The order of resistance to L. major infections from most resistant to most susceptible strain of mouse is: 1) CBA, 2) C57BL/6, 3) DBA/2 and BALB/c [1]. This is exactly mirroring their respective risks to succumb to obesity/type II diabetes-associated pathology such as atherosclerosis: 1) CBA, 2) C57BL/6, 3) DBA/2 and BALB/c (according to the Jackson laboratory’s website: http://jaxmice.jax.org/info/ready.html, http://jaxmice.jax.org/strain/000654_2.html, http://jaxmice.jax.org/strain/000651.html, http://www.informatics.jax.org/external/festing/mouse/docs/DBA.shtml ). It is also worth noting that males seem to have an increased risk to develop atherosclerosis/type II diabetes/obesity whilst being slightly better at coping with Leishmania infections.

2) Leishmaniases are diseases affecting the young particularly harshly (in mice as well as in man, resistance seems to increase with age, or expressed another way the risk of disease drops with age) [48], whereas the risk of developing type II diabetes mellitus increases the older mice or humans get [49;50]. It was recently shown that SOCS expression increases in lymphocytes and granulocytes as well as the rat hypothalamus a consequence of ageing [51-54]. However, this phenomenon has not been shown directly for macrophages yet.

3) It was shown that BALB/c mice remained susceptible to certain infections with certain subtypes of L. major even when the major Th2 cytokine IL‑4 was knocked out [2]. This was probably due to other cytokines such as IL‑13 and IL‑6 being able to take over some of the functions performed by IL‑4 under normal conditions and to induce arginase in infected and bystander macrophages [55].

4) It was observed that there is an early immune response to L. major in susceptible mice producing an early wave of IL‑4 the intensity and kinetics of which suggested a recall/memory response of pre-existing T cells rather than freshly primed and differentiated T lymphocytes [56;57]. A T cell population with a specific TCRαβ usage was shown to react to Leishmania LACK antigen and to be the source of the early wave of IL‑4. The population probably arose primed by gut antigen and deletion of these T cells prevented early IL‑4 release and prevented the development of non-healing lesions [58-60]. However, it was shown that early IL‑4 could be detected equally in resistant mice such as C57BL/6 and therefore did not predict the susceptibility to Leishmania infections [61]. This argues for an active switch from Th2 response to Th1 response that is not being carried out in BALB/c mice.

5) Whilst it has often been said that BALB/c are generally more Th2-prone than C57BL/6 mice and Th2 responses have been shown to be crucial to resistance against the filarial nematode Litomosoides sigmodontis, BALB/c are susceptible to the infection while C57BL/6 are resistant [62;63]. Again gender seemed to have an effect on the outcome of the disease, with higher protection observed in males [62]. Mirroring results in L. major infection, BALB/c mice deficient in IL‑4 remained susceptible [63]. However, IL‑4 is required to prevent filarial nematode development in resistant mice, arguing that IL‑4 is necessary but not sufficient and that the nematode can develop in susceptible mice in-spite of IL‑4 present.

6) TLR have been implicated in the response to Leishmania either directly in vitro (TLR2 recognising Leishmania LPG und resulting in SOCS expression, TLR3 recognising unidentified PAMP in Leishmania which may be gRNA double-stranded RNA) [64;65] or indirectly in vivo (TLR4-deficient mice on a resistant background controlling the disease less efficiently: possible TLR4 activators: hyaluronan, heat-shock proteins, microbial PAMP due to unintentional co-infection, or as–yet unidentified Leishmania PAMP [66], involvement of MyD88 [64;67;68]).

7) Infection of mice on the BALB/c background with L. mexicana and promastigote-secreted gel (PSG) gave following results. Addition of PSG to infections of wild-type BALB/c exacerbated the course of the disease. Adding PSG to infections of IL‑4-deficient BALB/c was beneficial to the mice for a short while (smaller lesions) but lesions were nonhealing all the same. Adding PSG to infections of BALB/c mice deficient in T1/ST2 showed no significant difference to the courses of infection in the absence of PSG (Matthew Rogers, personal communication). I interpret these results in such a way that PSG multiplies the effects of the prevailing cytokine environment in an at least partially T1/ST2-dependent way.









8) T1/ST2 is the receptor for IL‑33 [69-71] and has been shown to be critical for LPS tolerance in vivo, acting as a negative regulator of TLR signalling [72;73], possibly functioning by inducing SOCS expression.

9) The current method to treat leishmaniases is heavily reliant on preparations of the heavy metal antimony (Sb, drugs: pentostam, stiboglucanate) a member of the same group of elements as arsenic (As). (see: "http://upload.wikimedia.org/wikipedia/commons/thumb/f/fe/Circular_form_of_periodic_table.svg/400px-Circular_form_of_periodic_table.svg.png")

The mechanism of action is not entirely clear although or (paradoxically perhaps) because antimony had been used as a medicine since antiquity and in its modern preparation since the first half of the 20^th century. Based on observations in responses to helminthic infections, it had been speculated that antimony could interfere with the sugar metabolism of the parasite [74], but promastigotes do seem to be able to survive culture in the presence of antimony. Apart from applications in anti-leishmanial chemotherapy very little is know about the pharmacological properties of antimony. However, arsenic, its smaller and more famous cousin, has been used in a number of pharmacological studies. It ranks highly in the lists of toxic agents and chronic low-dose arsenic exposure has been linked to a number of cancers. However, the mechanism of carcinogenesis is not fully understood yet. It is a known carcinogen yet it is not a potent mutagen in itself. The lack of any prominent signal transduction pathway and animal model for arsenic carcinogenesis has led to the belief that it acts as an epigenetic carcinogen. It was only recently shown that long-term low-dose exposure of arsenic leads to a reduction in intracellular S-adenosylmethionine (SAM) and a loss of DNA methylation [75]. This is most likely due to methylation of arsenic depleting intracellular levels of SAM and an observed repression of DNA methyltransferases DNMT1 and DNMT3A gene expression. (see: "www.ehponline.org/members/2005/8600/8600.html" and "www-ermm.cbcu.cam.ac.uk")


Model

The model I propose consists of the following elements:

Parasite survival is dependant on the macrophage actively providing Leishmania with nutrients. One, if not the main, pathway for the parasite to obtain nutrients is via arginase which acts in competition with iNOS in mouse macrophages. (Arginine depletion in the environment as a result of increased arginase activity further exacerbates the disease by preventing T cells from functioning properly.)

It is not so much the cytotoxic activity of NO radicals that prove fatal to the parasite, but the inhibition of arginase. Arginase and iNOS compete not only for the same substrate, but iNOS produces an inhibitor of arginase (NOHA) as an intermediate product of its catalysis of arginine to citrulline and NO. In addition, the two enzymes are induced by cytokines that have inhibitory/regulatory effects on the other cytokine (IFN‑γ induces iNOS, IL‑4 induces arginase, IFN‑γ inhibits effects of IL‑4 signalling and vice versa). NO leads to TNF‑α expression and therefore indirectly to increased SOCS expression, adding another layer of regulation to this network. IFN‑γ signalling leads to STAT1 phosphorylation and activation, which drives transcription of IFN‑γ-responsive genes, including SOCS1 and SOCS3, in macrophages. IL‑4 signalling most commonly involves phosphorylation and activation of STAT6, leading to increased expression of genes such as of SOCS1 and SOCS2 in macrophages. The different SOCS have different affinities for their target proteins, at limiting concentrations SOCS may therefore inhibit one type of cell signalling, but at higher concentrations inhibit additional signalling pathways that were only “second choice”. Cells may thus limit their ability to be affected by stimuli arriving after the first cell signalling event in a highly receptor-specific way and integrate various signals to produce a qualitatively nuanced response.

The main point for macrophages and the immune system seems to be overcoming the initial IL‑4 response of pre-primed T cells and shut down the signalling pathways leading to arginase expression. This can happen either by TLR activation or other mechanisms of innate immunity. Difference in SOCS expression in macrophages would be an obvious choice to look at. However, SOCS expression in macrophages might be downstream of differential SOCS expression in T cells. Once the adequate kind of SOCS expression has been established in the host cells (type of SOCS, quantity, timing, location, resulting from and affecting the immune response via various ways of regulation and feedback) the cells do not receive the orders to produce arginase and stop to provide the parasite with nutrients. The result is partly direct killing of parasites by cytotoxic mechanisms (radicals, etc), but perhaps even more importantly parasitic “death by neglect” (albeit an active, highly regulated and maintained form of neglect). This hypothesis is supported by the observation that SOCS1-deficient or impaired (heterozygous) mice show increased susceptibility to L. major infection [76] and L. donovani, L. amazonensis and L. major all induce various amounts of SOCS3 [77]. The expression of SOCS3 had been interpreted by the authors as a possible mechanism of suppression of activation, however the difference between classical and alternative activation had not been addressed. The authors seemed to suggest that SOCS3 expression may have beneficial effects on parasite survival, which might very well be the case in a Th1 environment, but under Th2 conditions SOCS activity might lead to the opposite outcome. De Veer and colleagues also showed TLR-induced SOCS3 expression, but I am unaware of any work following on from this demonstrating host-protective, parasite-killing effects of SOCS3 [64] which leaves the role of SOCS3 unresolved.

There are various ways that might explain why BALB/c might be less adapt at shutting off certain cytokine signals, but the most promising target to investigate seems to me to look at hypermethylation of SOCS (promoter) sequences.

SOCS proteins act as negative regulators to a great number of cell signalling processes including IL‑4R and insulin receptor signalling. The observed correlation between resistance to Leishmania infections (advantageous to the mouse) and insulin resistance (pathologic) in different strains of mice seems to suggest SOCS as the common link. Not only do they inhibit physical association of receptor and adapter proteins by occupying crucial pY residues, their SOCS box domains can act as ubiquitin-E3-ligases promoting the mono-, multi- or poly-ubiquitination of target proteins, resulting in down-regulation of trans-membrane receptors through internalisation, redistribution of membrane bound proteins to endosomal/lysosomal compartments (TLR [78]) or proteasomal degradation of cytosolic proteins (possibly STAT, other SOCS proteins [79]). The SOCS-induced shuttling of TLR to the endosomal/lysosomal pathway could be used in antigen-presenting cells as an efficient way to redirect microbial material to cellular compartments that then ensure preferential/efficient antigen presentation (as demonstrated in the case of Toxoplasma gondii and TLR11 [78]).

DNA methylation patterns develop over time and often are tissue- or cell-specific. In mammalian cells, DNA methyltransferases use SAM to preferentially methylate CG islands of genomic DNA. Maintenance methyltransferases ensure that methylation patterns are preserved and passed on to following generations of cells or organisms. Various cancers show hypermethylation of SOCS genes which leads to reduced SOCS-mediated signal inhibition and increased activational stimuli that the aberrant cancer cell line can use to proliferate and spread. Two compounds acting as DNA methylation inhibitors have recently been approved for treatment of certain cancers (e.g. 5-aza-2'-deoxycytidine) that may be especially effective in cancers reliant on activational/differentiation stimuli that have become reassigned growth and proliferative roles in cancer cells.

The fact that antimony’s homolog arsenic seems to function as an inhibitor of DNA methyltransferases can be exploited in the search for new treatments of leishmaniases. In theory, if SOCS proteins are indeed involved in the control of leishmaniases and are down-regulated because of DNA hypermethylation, then treatment with DNA hypomethylating agents such as 5-aza-2'-deoxycytidine might de-repress SOCS expression. SOCS proteins could then be efficiently expressed and could thus shut down the parasite’s life support system, by inhibiting IL‑4/IL‑13/IL‑10/IL‑6-driven arginase expression. I suggest that 5-aza-2'-deoxycytidine could be a prime drug candidate used in treating leishmaniases if it were possible to target the delivery of the molecule to the host cells. It is more efficient in demethylating DNA and functions in a different way to heavy metals such as antimony and arsenic which can be deactivated by leishmanial arsenate/antimonate reductases. In fact antimony-resistance of Leishmania is a serious and increasing problem that could be solved by switching to non-heavy metal-based drugs such as 5-aza-2'-deoxycytidine. Vice versa, antimony-based drugs might prove beneficial to treating certain forms of cancer. However, uptake into target cells, side effects and other issues such as costs have not been taken into account at this point. While the model offers a rational explanation for the treatment of leishmaniases, and brings together a number of areas of basic cell biology, immunology and parasitology, it remains highly speculative.

However, the rewards might outweigh the risks. The new drug candidate for treating leishmaniasis edelfosine looks to me like a potential inhibitor of DNA methylation (possibly inhibiting DNMT via its E18-O-CH3 group) and the observed effects of the drug on cell biology (vacuoles, uncoordinated cellular transportation and architecture) seem to imply consistency with the proposed upregulation of SOCS molecules resulting in ubiquitination and relocalisation of proteins and membranes to endosomal/lysosomal compartments.

In addition, the model does allow for a number of predictions that can be tested.

A) Are SOCS mRNA/proteins differentially expressed in mice / different cell types in mice? (SOCS3 in macrophages? (involved in downregulating cytokine signalling of IL‑4, IL‑13 and IL‑6), whereas SOCS2 has been shown to downregulate SOCS3 levels via ubiquitination [79] and would therefore be considered rather a negative influence on the control of Leishmania infections (in line with its association and induction by Th2 cytokine responses), SOCS5 is another potential candidate mediating protective effects, but there is more evidence suggesting that SOCS3 might be crucial).

B) Are there differences in methylation status of SOCS genes in different strains of mice / different cell types in mice? (Methylation specific real-time PCR analysis could be used to detect differences in gene-specific DNA methylation [80])

C) Are gender differences in susceptibility to certain diseases attributable to SOCS expression/ SAM content/ DNA methylation status?

D) Does antimony treatment result in a changed methylation profile of SOCS genes?

E) Is SAM content/ DNA methylation linked to pathogenesis of Leishmania in visceral leishmaniases?

F) Does 5-aza-2'-deoxycytidine act as an anti-leishmanial drug? Is edelfosine a demethylating agent and does it induce SOCS expression? (Complications due to TLR9-mediated recognition of unmethylated CpG need to be considered even if at one time it was very much debated whether TLR9 indeed recognises unmethylated CpG sequences or naked, single-stranded DNA instead. In the case of antimony treatment in patients and mice TLR9 activation may even be beneficial to resolving the disease (uptake of antimony via Glut1 transporter into infected macrophages [81], CpG is generated and activates TLR9, which then sets off a response characterised by IFN‑γ helping to shut down the arginase activity)).

G) Does the model explain complications in leishmaniasis such as post-kala azar dermal leishmaniasis? (Withdrawal of antimony may result in increased DNA methylation over time, affecting SOCS expression, host cells with latent infection receive instructions to increase arginase activity, resurgence of the Leishmania in areas that have been reached by latently infected cells….)

H) What would be the phenotype of mice derived from resistant (CBA, C57BL/6) mice with an IL‑4R that did not allow for inhibition by SOCS be regarding susceptibility to L. major?

I) Similarly, can this model be applied to L. sigmodontis infections in mice?

J) Does PSG lead to a reduction in SOCS expression?

K) Is T1/ST2 involved in the regulation of SOCS expression? (Implications for transplantation immunology, because of the link between mast cells expressing T1/ST2 and Treg. Possible links to LPS tolerance – treatments for sepsis (antimony-based, etc – compare to the effects of lead on LPS tolerance)).

L) Are SOCS responsible for/involved in targeting membrane TLR to the endosomal/lysosomal compartment upon TLR activation via receptor ubiquitination? (demethylating agents could lead to SOCS expression and deregulation of ubiquitin/sumo/etc-mediated intracellular trafficking.)

If all or at least some of this is found to be true, it might offer an advancement in our understanding of the way the parasite and the host cell interact and might help in the development of potential drug candidates. The possibility that DNA methylation might determine the biological age of organisms and therefore 6-8-week-old mice of different strains might have aged in different ways is quite an exciting concept. The potential of this idea seemed so big to me at the time that I could not help myself and tried to talk to people about it who were not interested. The hard part is getting experimental proof for all this theorising. Anyone who finds these ideas useful is welcome to "steal" them and to see if they are of any use in the real world. The role of SAM in epigenetics such as DNA methylation and histone modification is intriguing, but may not be the main or only role to consider. SAM also plays a very important role in polyamine synthesis, which might be a different line of research worth pursuing, especially given the role of spermidine and trypanothione.

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