Introduction
Over evolution, some amino-acid catabolic pathways have become critical checkpoints
in immunity (1–3). The associated immunoregulatory effects rely on the depletion of
specific amino acids in the microenvironment and/or generation of biologically active
metabolites (4). Consumption of l-arginine (Arg) by arginase 1 (ARG1) represents a
well-known immunoregulatory mechanism exploited by M2 macrophages (5) and myeloid-derived
suppressor cells (MDSCs) (6–8) in tumor settings. ARG1 is also expressed by human
neutrophils (9). Indoleamine 2,3-dioxygenase 1 (IDO1)—a powerful immunosuppressive
enzyme catalyzing the first, rate limiting step in l-tryptophan (Trp) catabolism—depletes
Trp and produces immunoregulatory molecules collectively known as kynurenines (10–13).
High IDO1 expression and catalytic activity occur in dendritic cells (DCs)—professional
antigen presenting cells—in response to interferon-γ (IFN-γ) (8, 10, 11). Unlike ARG1,
IDO1 is also endowed with non-enzymatic signaling activity in DCs that, in the presence
of transforming growth factor-β (TGF-β) in microenvironments, leads to durable immunoregulatory
effects (14, 15). In conventional DCs (cDCs), a relay pathway—marked by the sequential
activation of ARG1 and IDO1—promotes a potent immunoregulatory phenotype (8, 16, 17).
In this setting, spermidine, i.e., a polyamine produced downstream of the ARG1-dependent
pathway (18), is capable of triggering IDO1 phosphorylation and signaling, and thus
may represent the critical molecular interconnection between the two enzymes (8, 16).
Here, we discuss the possible protective vs. pathogenetic roles of the interplay between
IDO1 and ARG1 in reprogramming immune cell functions in neoplasia and autoimmune diseases.
The ARG1 and IDO1 Interplay as Physiologic Immune Checkpoint
As all biological processes, immune responses rely on both energy-consuming and energy-producing
pathways (19). The availability of specific substances and the immunological signature
of the microenvironment directly control immune cell fate and functions. Pathogen-associated
molecular pattern (PAMPs) and damage-associated molecular pattern (DAMPs) molecules
(recognized by pattern recognition receptors such as Toll-like receptors or TLRs)
as well as amino acids, glucose, and fatty acids drive T-cell proliferation. Indeed,
among immune cells, T lymphocytes are particularly dependent on nutrient availability
and such feature (known as auxotrophy) has evolved as biological containment strategy
that promotes the life-or-death decision (19).
By reducing the supply of indispensable amino acids, IDO1 and ARG1 directly suppress
T cell proliferation and differentiation. The inadequacy of Arg and Trp substrates
promotes a state of quiescence, whereby non-essential functions are temporarily quenched,
including the cell cycle progression in the G0-G1 phase and the expression/activation
of the TCR ζ-chain (2, 20, 21). IDO1 and ARG1 are indeed considered as physiological
checkpoints ensuring a short-lived immunosuppression in normal pregnancies. In the
placenta, DCs and extravillous trophoblasts highly expressing IDO1 and ARG1 secure
a reversible T cell hyporesponsiveness and thus the survival of the fetus in utero
(22, 23).
The activity of ARG1 and IDO1 translates not only into amino acid deprivation, but
also in the production of metabolites endowed with several physiologic effects. l-kynurenine
and spermidine, derived from Trp and Arg, respectively, are clear archetypes of non-inert
byproducts that can influence immune and non-immune cell functions. In particular,
l-kynurenine, by engagement of the aryl hydrocarbon receptor (AhR; a ligand-activated
transcription factor), favors the differentiation of regulatory T (Treg) cells and
induces IDO1 expression in DCs (24). On the other hand, the polycationic spermidine
regulates cell growth and proliferation, and it affects several signal transducing
pathways by interacting with ion channels, membrane receptors, and kinases (18).
Under specific conditions (as those dominated by TGF-β), Arg and Trp metabolic pathways
are co-activated, thus potentiating the immunoregulatory phenotype of DCs and MDSCs
(8, 25). The intimate relationship between ARG1 and IDO1 is allowed by spermidine,
which activates the non-enzymatic functions of IDO1 and thus reprograms the cDC toward
a long-term, immunoregulatory phenotype. More specifically, through Src kinase activation,
spermidine induces the phosphorylation of IDO1, which, in turn, behaves as signaling
molecule, promoting activation of the non-canonical pathway of NF-κB and induction
of TGF-β1 and IDO1 expressions (3, 8). Contrary to spermidine, the small molecule
nitric oxide (NO; derived from the Arg breakdown catalyzed by NO synthase) negatively
regulates Trp metabolism, as it directly binds the heme prosthetic group and thus
blunts the enzymatic function of IDO1 (26). However, besides this effect that would
dampen IDO1-mediated immunosuppression, high levels of NO can combine with superoxide
anion thus generating reactive nitrogen species that compromise both the activity
and migration of T cells at the tumor site (27). Of note, it has been recently shown
that AhR can sustain intracellular polyamines production at least in neoplastic conditions
(28). However, whether such positive modulation belongs to a physiologic, bi-directional
regulation program, where Trp metabolites and/or IDO1 itself affect ARG1 functions,
has not been investigated yet.
ARG1 and IDO1 in Neoplasia
Difference in the metabolism of normal and cancer cells underlie the quest for more
specific and less toxic therapies than those currently used. Tumor development is
conditioned by genetic changes in malignant cells, immunological tolerance, and immunosuppression
(29). At the initial stages of carcinogenesis, the immune system is capable of anti-tumor
activity; however, cancer progression compromises the action of T helper type 1 (Th1)/Th2/Th17
lymphocytes via Treg cells, tumor-associated macrophages (TAMs), and MDSCs, resulting
in immunosuppression and loss of reactivity to tumor antigens (30, 31). Recently,
much attention has been dedicated to the influence of Arg and Trp metabolic pathways
on both tumor cell growth and host's immune antitumor response. Arg is essential for
the maturation of the TCR ζ-chain, and its deprivation impairs T cell ability to activate
tumor immunity. MDSCs deplete Arg because they express high levels of ARG1, and their
number increases 4–10 times depending on the type of cancer. For these reasons, in
cancer immunotherapy studies, the effects of both deprivation and supplementation
of Arg have been tested, the former on the assumption that tumors may be Arg auxotrophic,
and the latter in an effort to counteract the detrimental effect of ARG1-competent,
tumor-associated MDSCs on the host antitumor response. Overall, seemingly contradictory
results were found in such oncological therapies based on the deprivation or supplementation
of Arg, and those results are not easily reconciled (29). In particular, the high
efficacy of subtracting Arg to Arg auxotrophic tumors may hardly explain per se the
global protective effect of this maneuver, in that most tumors may ultimately activate
the arginine-succinate synthetase (ASS1) pathway that enables synthesis of Arg from
citrulline. The recent finding of a supportive influence of ARG1 on IDO1-dependent
tolerogenesis (8)—which would impair host antitumor responses—suggests that it is
not the Arg subtraction to the tumor that matters so much as the impairment of ARG1's
supportive role in allowing full expression of the IDO1 mechanism in suppressing antitumor
responses. In fact, ARG1+ MDSCs, obtained by cell incubation with medium derived from
mouse melanoma cells, can condition DCs to acquire an IDO1-dependent, immunoregulatory
phenotype in vivo via production of polyamines (8, 16). Therefore, these data would
sustain the existence of an immunosuppressive cross-talk mechanism between distinct
cells present in tumor stroma and expressing ARG1 and/or IDO1 (32). The mechanisms
whereby IDO1 acts as an immunosuppressant are multiple, and they are detailed elsewhere
(2, 10, 11, 33).
There are, however, clinical settings where pharmacological administration of Arg
resulted in cytoreductive effects in patients with Arg non-auxotrophic tumors (29).
Paradoxical as it seems, this effect could again be explained by the relationship
between ARG1 and IDO1 in immune cells. Increased ARG1 activity might lead to IDO1-dependent
Trp starvation in cancer cells. Because Trp is an essential and the rarest of all
amino acids, this likely results in an overall proteostatic action that affects fast-growing
tumors, as discussed elsewhere in detail (3).
With specific regard to Arg auxotrophy, this phenomenon takes place in certain tumors
and is caused by the silencing of ASS1 or arginine lyase genes. Those tumors are characterized
by an intrinsic chemoresistance and thus a poor prognosis. Nevertheless, on a positive
note, Arg auxotrophy theoretically favors the treatment of these tumors with Arg-degrading
enzymes. Among the most frequently applied Arg-degrading agents are arginine deiminases
(ADI) from bacteria. The antitumor effects of ADI derived from different bacteria
have been extensively studied in vitro and in vivo [for review, see (34)]. Mycoplasma-derived
ADI-PEG20 is the one most commonly used and is under clinical investigation as a single
agent therapeutic as well as in combination with other chemotherapeutic compounds.
Mechanistically, ADI reduces metabolic activity in tumor cells, contributing to autophagy,
senescence, and apoptosis in Arg auxotrophic cells (34). Although clinical trials
are promising, the development of resistance after initial treatment is challenging,
as illustrated above. Furthermore, an ADI interference within the tumor microenvironment
is to be considered. Again, non-specific subtraction of the substrate for ARG1 may
indirectly affect the host response to the tumor via effects on IDO1.
Another important issue in cancer is the expression of Arg and Trp transporters in
tumor and immune cells. Among Arg carriers (cationic amino acid transporters or CATs),
the most important appear to be CAT1, which is constitutively expressed in several
tissues, and CAT2B, normally inducible by inflammatory cytokines (35). CAT1 is often
overexpressed by tumor cells, and event that can favor tumor growth. In an experimental
model of prostate cancer, CAT2B, which allows a rapid transport of Arg into the cell,
is expressed at higher levels in tumor-infiltrating as compared to peripheral MDSCs
(36). Moreover, the upregulation of CAT2 is coordinated with the induction of both
NOS2 and ARG1, thus further favoring Arg uptake by MDSCs at the tumor site. Subsets
of human melanoma cells are also characterized by very high levels of CAT2B expression,
possibly due to the secretion of inflammatory mediators by the tumor cells themselves
(35). Overexpression of Trp carriers (mainly, LAT1/CD98 and SLC6A14) is also involved
in the increased proliferation and chemoresistance of several tumor cell types (37).
Because SLC6A14 is a broad specific amino acid transporter that can also transfer
Arg and its expression can be upregulated by IDO1 (by a mechanism not identified yet)
(38), the “doors” for the cell entrance of Arg and Trp may represent suitable cancer
drug targets capable of interfering with both ARG1 and IDO1 pathways (39).
Therefore, new insight is definitely needed into the molecular mechanisms underlying
the antitumor effects of Arg starvation in both host and tumor, which might facilitate
the refinement of IDO1 inhibitory approaches in cancer immunotherapy.
ARG1 and IDO1 in Autoimmunity
The use of checkpoint inhibitors in tumor immunotherapy is frequently accompanied
by the development of autoimmune diseases (40), suggesting that the exploitation of
immune checkpoint molecules could be a valid therapeutic means in autoimmunity (4,
41). Because both ARG1 and IDO1 act as immune checkpoint mechanisms in neoplasia,
their functional “alliance” in specific immune cells could be remarkably effective
in controlling adaptive immunity toward auto-antigens.
IDO1 is defective in DCs of non-obese diabetic (NOD) mice (42), an experimental model
of human autoimmune diabetes (type 1 diabetes or T1D), and maneuvers aimed at enhancing
its expression and activity will exert therapeutic effects in prediabetic and also
overtly diabetic animals (43, 44). In T1D patients, a significantly reduced IDO1 expression
can be observed in peripheral blood mononuclear cells (PBMCs) (17) and in pancreatic
β cells (45), normally producing insulin. In PBMCs, the defect can be corrected by
tocilizumab, a blocker of the interleukin 6 (IL-6) receptor, which inhibits the IL-6–dependent,
IDO1 proteasomal degradation (17). In T1D, although its expression and function in
immune cells remains unclear, endothelial ARG1 induces the vascular dysfunction associated
with hyperglycemia (46). Moreover, administration of difluoromethylornithine (DFMO),
a potent inhibitor of polyamine production, protects NOD mice from the development
of diabetes.
In experimental models of rheumatoid arthritis (RA), an inflammatory/autoimmune disease
of the capsule surrounding joints, lack of IDO1 expression reduces the time to develop
a more severe disease (47). Moreover, the protective effects of interferon-α rely
on the activation of a TGF-β/IDO1 axis in plasmacytoid DCs (48). Although ARG1+ M2
macrophages contribute to resolve arthritis inflammation in mice (49), ARG1 activity
may be responsible for subclinical endothelial dysfunction also in RA patients (50).
Interestingly, methotrexate, an immunosuppressive drug widely used in RA, greatly
inhibits the synthesis of polyamines in lymphocytes of RA patients (51).
A definitely clearer picture is emerging in autoimmune neuroinflammation. Administration
of 3-hydroxyanthranilic acid (3-HAA; a Trp metabolite of the kynurenine pathway) (52)
or of an orally active synthetic derivative thereof (53) ameliorates neuroinflammation
and paralysis in mice with acute experimental autoimmune encephalomyelitis (EAE),
a model for multiple sclerosis (MS). Moreover, 3-HAA–treated DCs express higher levels
of TGF-β and induce the generation of Treg cells (52). Conversely, administration
of 1-methyltryptophan (1-MT), a standard inhibitor of IDO1, exacerbates the clinical
course of EAE (54, 55). In leukocytes infiltrating the spinal cord of untreated mice,
IDO1-expressing cells exhibit the same morphology as activated macrophages/microglia
(54). VCE-004.8, a semisynthetic cannabinoid, protects from EAE, possibly by upregulating
ARG1 in macrophages and microglia (56). A LewisX trisaccharide of schistosome eggs
reduces EAE severity by a TLR-mediated mechanism that enhances both ARG1 and IDO1
expression in CD11b+Ly-6Chi inflammatory monocytes (57). Expression and activity of
ARG1 and IDO1 are significantly reduced in PBMCs from MS patients as compared to healthy
control subjects (58). Spermidine, the polyamine produced downstream ARG1, protects
from autoimmune-directed demyelination of neurons in acute EAE (59). The effect appears
to be related to an immunosuppressive function acquired by ARG1+ macrophages, since
(i) their depletion or the administration of an ARG1 inhibitor abolishes spermidine
therapeutic activity in vivo and (ii) the polyamine induces ARG1 in macrophages (59).
Therefore, although in both T1D and RA the pathways of Arg and Trp metabolism do not
seem to be properly interlinked (and this may require cautions when attempting immunotherapies
potentiating both ARG1 and IDO1), in MS, the pieces of evidence, when put together,
would suggest that the induction of the immunosuppressive interplay between ARG1 and
IDO1 would represent a valid therapeutic objective.
Conclusions and Perspectives
In neoplasia, both ARG1 and IDO1 are often overexpressed, either singly in tumor cells
themselves (IDO1) or in association (i.e., both enzymes) in MDSCs and DCs, and they
contribute to the impairment of the host anti-tumor immunity. However, the effect
of Arg starvation on tumor cells may dampen their proliferation and therefore ARG1
inhibition as therapeutic strategy may have some caveats (Figure 1). In the majority
of autoimmune disorders, the bulk of data would suggest that IDO1, expressed by either
DCs or macrophages, stands out as an effective immune checkpoint molecule. In contrast,
more often than not, ARG1 appears to be more pathogenetic than protective, possibly
owing to the enzyme capacity to subtract Arg for NO production, which can be necessary
for the resolution of damages induced by autoimmunity (4, 60). However, in autoimmune
neuroinflammation, the available cues would indicate that both ARG1 and IDO1, expressed
by macrophages and/or DCs, act as immune checkpoint molecules in EAE and that spermidine,
i.e., the molecular connection between the two enzymes in a physiologic setting (8),
exerts significant therapeutic effects on its own. Therefore, further investigations
on Arg metabolism in neoplasia and autoimmune disorders and its possible cross-talk
with IDO1 are needed for a full understanding of its role, protective vs. pathogenetic.
Figure 1
The role of ARG1 and IDO1 in neoplasia and autoimmunity. The up-regulation of ARG1
activity, induced by the cytokine TGF-β, transforms l-arginine (l-Arg) into l-ornithine
(l-Orn), which is further metabolized by ornithine decarboxylase (ODC) into polyamines
(PUT, putrescine; SPD, spermidine; and SPM, spermine). SPD, through the activation
of the Src kinase, promotes the phosphorylation of IDO1 and thus favors the initiation
of immunoregulatory signaling events in DCs. Once phosphorylated, IDO1 recruits tyrosine
phosphatases (SHPs) and promotes a signaling pathway that upregulates the expression
of genes coding for IDO1 and TGF-β, thus creating a self-sustaining circuitry responsible
for the maintenance of immune tolerance over the long-term. Moreover, IDO1 catalyzes
the conversion of l-tryptophan (l-Trp) into l-kynurenine (l-Kyn), which activates
the aryl hydrocarbon receptor (AhR). AhR further induces IDO1 expression in DCs and
sustains the production of polyamines by up-regulating ODC. Whereas the pathogenetic
and protective role of TGF-β, SPD, and IDO1 in neoplasia and autoimmunity, respectively,
have been demonstrated, the role of ARG1 has been unclear and would require further
investigations. Gray arrows indicate the pathogenetic effects of IDO1, ARG1, SPD,
and TGF-β1 receptor signaling in neoplasia and brown arrows indicate the putative
protective effects of IDO1, SPD, ARG1, and TGF-β1 receptor signaling in autoimmune
diseases. Dotted lines are for molecules whose role is still unclear.
Author Contributions
GM, AI, MA, PP, and UG equally contributed in writing the manuscript. UG supervised
the final form.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.