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Abstract

Muramyl peptides (MPs) represent the building blocks of bacterial peptidoglycan, a critical component of bacterial cell walls. MPs are well characterized for their immunomodulatory properties, and numerous studies have delineated the role of MPs or synthetic MP analogs in host defense, adjuvanticity and inflammation. More recently, Nod1 and Nod2 have been identified as the host sensors for specific MPs, and, in particular, Nod2 was shown to detect muramyl dipeptide (MDP), a MP found in both Gram-positive and Gram-negative bacterial cell walls. Because mutations in Nod2 are associated with the etiology of Crohn’s disease, there is a need to identify synthetic MP analogs that could potentiate Nod2-dependent immunity. Here, we analyzed the Nod2-activating property of 36 MP analogs that had been tested previously for their adjuvanticity and anti-infectious activity. Using a luciferase-based screen, we demonstrate that addition of a methyl group to the second amino acid of MDP generates a MDP derivative with enhanced Nod2-activating capacity. We further validated these results in murine macrophages, human dendritic cells and in vivo. These results offer a basis for the rational development of synthetic MPs that could be used in the treatment of inflammatory disorders that have been associated with Nod2 dysfunction, such as Crohn’s disease.

Keywords

Nod2 muramyl peptides muramyl dipeptide adjuvant innate immunity inflammation

Introduction

Peptidoglycan (PG) is a critical component of the cell wall of Gram-positive and Gram-negative bacteria. The PG heterogeneous polymer is composed of a sugar backbone comprised of alternating N-acetylglucosamine and N-acetylmuramic acid (MurNAc) residues. A peptide chain is attached to the MurNAc sugar, forming a muramyl peptide (MP), and these stem oligopeptides can be crosslinked to form the three-dimensional lattice structure of PG. The capacity of polymeric PG, as well as MP residues [and, in particular, muramyl dipeptide (MDP)], to induce both inflammatory and adaptive immune responses has been studied for decades.¹ Indeed, a wealth of scientific reports from the past four decades examine how chemical modifications of MDP would affect the ability of this compound to enhance host clearance of bacterial pathogens or function as an adjuvant for antigen-specific immune responses.²

Following these studies on the immune properties of MPs, Nod1 and Nod2 were more recently identified as the critical cytosolic pattern recognition receptors (PRRs) that sense specific MPs in mammals, resulting in the induction of cellular defense pathways dependent on NF-κB, mitogen-associated protein kinase and autophagy.³ Nod1 detects meso-diaminopimelic acid-containing MurNAc-tripeptide (Mur-TriDAP) found predominantly in Gram-negative bacteria,^4,5 whereas Nod2 detects MDP found in both Gram-negative and Gram-positive bacteria.^6,7 More detailed studies on the minimal structural requirements of the MP ligands needed for Nod1 or Nod2 activation revealed that the MurNAc sugar is not required for Nod1 activation as the d-Glu-meso-DAP dipeptide (iE-DAP) is sufficient for detection and innate immune activation by this PRR.^8,9 Nod2, however, can only be activated by muramyl dipeptides that have an intact MurNAc ring structure, and the sugar has to be attached to a dipeptide moiety (l-Ala-d-Glu or l-Ala-d-isoGln).⁹

Recently, Nod2 was shown to bind directly to its MDP,¹⁰ suggesting that this protein acts as a bona fide cytosolic receptor. It was also reported previously that an intact cellular endocytic pathway was critically required for the activation of both Nod1 and Nod2, indicating that cytosolic internalization of ligands was necessary for activation.^11,12 The discovery of di- and tripeptide transporters located at the plasma membrane that are needed to internalize Mur-TriDAP and MDP further reinforces the idea that binding of MP ligands to Nod1 and Nod2 is essential for the induction of NF-κB signaling by these proteins. Specifically, it was shown that the oligopeptide transporter hPepT1 (also known as SLC15A1) acts as a specific transporter for MDP,¹³ but not Nod1 ligands,¹⁴ whereas the peptide transporter SLC15A4, which is expressed in early endosomes, is required for Nod1 stimulation by MurNAc-TriDAP and iE-DAP.¹¹ Finally, hPepT2 (also known as SLC15A2) was also found to be involved in MP internalization.^15,16

Early studies have reported that the stimulatory capacity of MPs can be enhanced considerably by the addition of acyl chains of various lengths to the core of what are now known as the Nod1- and Nod2-activating ligands (i.e. iE-DAP or MDP respectively),^17,18 and this property likely correlates with the enhanced capacity of acylated MPs to enter host cells by endocytosis. In addition, earlier studies (mainly from the 1970s and 1980s) provided detailed characterization of the structure/function relation for MPs and, in particular, MPs derived from MDP. However, since the identification of Nod2 as the cellular MDP sensor little effort was made to revisit these earlier studies and test the compounds from the synthetic MP libraries directly for their Nod2 stimulatory capacity. Here, we initially screened the Nod2-dependent NF-κB activation capacity of a library of 36 chemical derivatives of MDP and identified MurNAc-l-Ala-d-Glu-OCH₃ [MDP(d-Glu²)-OCH₃], a synthetic MDP-derivative in which the terminal d-isoglutamine was replaced by the α-methyl ester of d-glutamic acid, as a novel compound that exhibited a lower activation threshold than MDP. Accordingly, this compound also induced more robust Nod2-dependent innate inflammatory responses both in vitro and in vivo. Together, we found MDP analogs with either lower or higher activation thresholds that could be used as starting points for the development of new therapeutics with immunomodulatory properties.

Materials and methods

Reagents

The 36 MDP-derivative compounds used in this study (Table 1) were collected from MP libraries previously synthesized and evaluated for their biological activity (adjuvanticity and anti-infectious activity).^19–23 Larger quantities of MurNAc-l-Ala-d-isoGln (MDP), MDP(d-Glu²)-OCH₃ and MurNAc-d-Val-d-isoGln [MDP(d-Val¹)] that were needed for in vitro and in vivo analyses were synthesized according to previously reported procedures.^20,22

Table 1.

List of tested muramyl dipeptide derivatives used in the current study. Structure information and working names are provided. Adjuvant refers to reported adjuvant capacity of the tested compounds,³¹ whereas anti-infectious refers to previously reported ability to enhance host immunity against the pathogen Klebsiella pneumoniae.³²

#	Structure	Compound name	Adjuvant	Anti-infectious	Ref.
1	MurNAc-l-Ala-d-isoGln-l-Lys	MDP-l-Lys	++	++	31,32
2	MurNAc-l-Ala-d-Gln-OCH₃	MDP(d-Gln²)-OCH₃	++	++	31,32
3	MurNAc-l-Ala-d-Glu-OCH₃	MDP(d-Glu²)-OCH₃	++	++	31,32
4	MurNAc-l-Ala-d-Glu	MDP(d-Glu²)	++	++	31,32
5	MurNAc-l-Ala-l-isoGln	MDP(l-isoGln²)	−	−	31,32
6	MurNAc-l-Ala-d-isoGln-OCH₃	MDP-OCH₃	++	++	31,32
7	MurNAc-l-Ala-d-isoGln-d-Ala	MDP-d-Ala	−	−/+	31,32
8	MurNAc-l-Ala-d-Gln-NH₂	MDP(d-Gln²)-NH₂	−/+	−	31,32
9	MurNAc-l-Ala-d-Glu(OCH₃)₂	Muradimetide	++	++	31,32
10	MurNAc-d-Ala-d-isoGln-d-Ala	MDP(d-Ala¹)-d-Ala	−	−	31,32
11	MurNAc-l-Ala-d-Gln-OnC₄H₉	Murabutide	++	++	31,32
12	MurNAc-l-Ala-d-isoGln	MDP	++	++	31,32
13	MurNAc-N-methyl-l-Ala-d-isoGln	MDP(N-Me-l-Ala¹)	+/−	−	31,32
14	MurNAc-d-Val-d-isoGln	MDP(d-Val¹)	+	−	31,32
15	MurNAc-d-Ser-d-isoGln	MDP(d-Ser¹)	+	−	31,32
16	MurNAc-l-Ser-d-isoGln	MDP(l-Ser¹)	+	−	31,32
17	MurNAc-Gly-d-isoGln	MDP(Gly¹)	−/+	−	31,32
18	MurNAc-d-Ala-d-isoGln	MDP(d-Ala¹)	−	−	31,32
19	MurNAc-l-Val-d-isoGln	MDP(l-Val¹)	+	−/+	31,32
20	MurNAc-l-Pro-d-isoGln	MDP(l-Pro¹)	+	−	31,32
21	MurNAc-N-methyl-d-Ala-d-isoGln	MDP(N-Me-d-Ala¹)	−	−	31,32
22	MurNAc-l-Thr-d-isoGln	MDP(l-Thr¹)	++	−/+	31,32
23	1-0-β-methyl-glycoside-MDP		−	−	^b
24	NorMurNAc-l-Ala-d-isoGln	Nor-MDP	++	+	33,34
25	1-0-α-methyl-glycoside-MDP		−	−	^b
26	MDP(d-Ser¹)-A–L		−	−	^b
27	6-0-Suc-MDP(d-Gln¹)-OnC₄H₉-A–L^a		++	++	19
28	MDP(d-Val¹)-A–L		−	−	^b
29	6-0-Suc-MDP(d-Gln¹)-OnC₄H₉		++	++	34
30	MDP-A–L		++	++	19
31	MDP(d-Ala¹)-A–L		−/+	−	19
32	4-0-Ac-6-0-Suc-MDP-OCH₃		++	++	34
33	l-Ala-d-isoGln	Dipeptide	−	−	33
34	MurNAc	Sugar	−	−	33
35	d-Ala-d-Gln-A–L		−	−	^b
36	l-Ala-d-isoGln-A–L		−	−	19

Suc, succinyl; A–L, multi-poly(dl-alanine)–poly(l-lysine) carrier.

Pierre Lefrancier and George Bahr, personal observations.

Luciferase assays

NF-κB activation luciferase assays in cells over-expressing Nod2 were performed as described previously.⁶ Briefly, HEK293T cells were transfected overnight with 30 ng of Nod2 and 75 ng of Igκ luciferase reporter plasmid. The cells were treated simultaneously with 1, 10, 100 or 1000 ng of the indicated muramyl peptide analog and the NF-κB-dependent luciferase activation was measured 24 h later on a luminometer.

Mice

C57BL/6 (Charles River) and Nod2^−/− mice were bred and housed under specific pathogen-free conditions at the Center for Cellular and Biomolecular Research, University of Toronto. The University of Toronto Animal Ethics Review Committee approved all animal experiments. Sex-matched mice that were 6–10 wk old were used for experiments.

In vivo cytokine response

C57BL/6 (Charles River) and Nod2^−/− mice were injected i.p. with the indicated dose of the MDP-derivatives, and serum was collected 2 h later. The concentration of the chemokine KC and the cytokine IL-6 in the serum was measured by ELISA (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol.

OVA-specific Ab response

WT and Nod2^−/− mice were immunized i.p. with a mixture of OVA (50 µg/mouse) and MDP, MDP(d-Val¹) or MDP(d-Glu²)-OCH₃ at the indicated concentrations (prime). The mice were re-injected with OVA 26 d later (boost). Blood from tail veins was collected 16 d later (42 d after the initial immunization) and sera of individual mice were analyzed. The levels of IgG1 in the serum were measured by sandwich ELISA.²⁴

Bone marrow-derived macrophage preparation

Bone marrow-derived macrophages (BMDM) were isolated from wild type (WT) and Nod2^−/− mice, and cultured as described previously.²⁵ Briefly, total bone marrow cells were seeded at 5 × 10⁶ cells in 10-cm dishes in 10 ml of DMEM [supplemented with 2 mM l-glutamine, 1 mM sodium pyruvate, nonessential amino acids (50 mM each) and 10% FCS supplemented with 30% of L cell-derived medium containing CSF activity]. On d 3, 10 ml of fresh medium was added to the cell culture. After 7 d of incubation, the non-adherent cells were removed and the remaining adherent cells were harvested by short incubation with ice-cold 1 × PBS. For experiments, BMDMs were seeded in complete culture medium in 24-well plates at 2 × 10⁶ cells/ml, pre-treated with cytochalasin D (1 µM) for 30 min and then stimulated with 50 µg/ml of MDP, MDP(d-Val¹) or MDP(d-Glu²)-OCH₃²⁵. Twenty-four h later, concentrations of KC and MIP2 were measured in the cell supernatants by ELISA.

Isolation of human dendritic cells

Monocytes were purified from human PBMCs of healthy donors using anti-CD14-conjugated magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Immature dendritic cells (DC) were generated by culturing monocytes in RPMI 1640 Glutamax medium [50 µM 2-mercaptoethanol, 10 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin (Invitrogen), and 10% FCS (Biowest, Nuaille, France) with 100 ng/ml GM-CSF (Miltenyi Biotec) and 40 ng/ml IL-4 (Miltenyi Biotec)] for 6 d. Immature DCs obtained were 100% CD1a⁺/CD14⁻.

Statistical analyses

Student’s t-tests or Mann–Whitney tests were performed using Prism (GraphPad Software, La Jolla, CA, USA), and P-values < 0.05 using a two-tailed 95% confidence interval were considered significant.

Results

In order to characterize the Nod2-dependent responses to synthetic MDP analogs, we used a library of 36 MDP derivative compounds (which includes MDP, compound 12) which had been tested previously for their adjuvanticity and anti-infectious activity, as summarized in Table 1. Interestingly, many of the tested compounds that had a modification at the second amino acid of MDP, such as MurNAc-l-Ala-d-Glu-OCH₃ [MDP(d-Glu²)-OCH₃], Murabutide and Muradimetide, had been shown to exhibit similar levels of enhanced protection against the bacterial pathogen Klebsiella pneumoniae and adjuvant capacity when compared to MDP (Table 1, compounds 1–12). This is in contrast to what was observed with MDP-derivatives modified at the first amino acid, such as MDP(d/l-Val¹), MDP(d/l-Ser¹) and MDP(l-Thr¹) (Table 1, compounds 13–22) which did not exhibit a protective effect against K. pneumoniae infection in vivo. However, many of these compounds were still reported to be functional adjuvants for the generation of specific Ab responses to antigens injected either by the i.v. or s.c. routes, although they demonstrated less effective adjuvanticity than MDP (Table 1).

We now know that Nod2 is the PRR mediating host sensing of MDP; thus, we were interested in assessing the capacity of the 36 MDP-derivatives to activate Nod2 at the cellular level. To do this we used the now well-established Nod2-Igκ luciferase reporter assay in HEK293T cells, and these cells were stimulated with our MDP-derivative library. Overall, our screen revealed, as expected, an excellent correlation between Nod2-stimulating capacities of these MDP derivatives and their previously assigned anti-infectious and adjuvant properties (Figures 1 and 2; Supplementary Figures 1–3; Table 1). Interestingly, we determined that the same second amino acid-modified MDP-derivatives that were shown to be good antibacterial compounds and adjuvants (Table 1) were also strong inducers of Nod2-dependent NF-κB activation in vitro (Figure 1). Interestingly, MDP(d-Glu²)-OCH₃ (compound 3), induced approximately fivefold more NF-κB activation than MDP when stimulated at the lowest dose of 1 ng/ml, indicating that MDP(d-Glu²)-OCH₃ has a lower activation threshold than MDP. Next, we determined that MDP-derivatives with the first amino acid of the d-configuration, such as MDP(d-Val¹) (compound 14), were compounds that could not induce Nod2-dependent NF-κB activation (Figure 2). MDP-derivatives modified at the anomeric function or d-lactoyl group of MurNAc (Table 1, compounds 23–25) exhibited reduced Nod2 stimulatory capacity compared to MDP (Supplementary Figure 1), while MDP compounds that were modified at two or more sites (Table 1, compounds 26–32) generally stimulated Nod2 less efficiently at lower doses (1, 10 and 100 ng) than MDP (Supplementary Figure 2). Finally, as described previously,⁹ MDP derivatives that lacked the MurNAc sugar or the dipeptide moiety were unable to stimulate Nod2 in vitro (Supplementary Figure 3), which reflected the inability of these compounds to act as antibacterials or adjuvants in vivo (Table 1, compounds 33–36).

Figure 1.

NF-κB stimulatory capacity of MDP-derivative compounds modified at the second amino acid. (A Structures of compounds 1–11 (see Table 1 for working names), which represent MDP derivatives modified at the second amino acid. Modifications with respect to MDP structure (compound 12) are written in red. (B) HEK293T cells that were transfected overnight with Nod2 plasmid and Igκ luciferase reporter plasmid were stimulated with 1, 10, 100 or 1000 ng of compounds 1–12, with 12 being MDP and acting as a positive control. The bar graphs represent the fold NF-kB activation over unstimulated cells. Values depict the average of three replicates from two independent experiments. Error bars depict SEM.

Figure 2.

NF-κB stimulatory capacity of MDP-derivative compounds modified at the first amino acid. (A) Structures of compounds 13–22 (see Table 1 for working names), which represent MDP derivatives modified at the first amino acid. Modifications with respect to MDP structure are written in red. (B) HEK293T cells that were transfected overnight with Nod2 plasmid and Igκ luciferase reporter plasmid were stimulated with 1, 10, 100 or 1000 ng of compounds 12–22, with 12 being MDP and acting as a positive control. Values depict the average of three replicates from two independent experiments. The bar graphs represent the fold NF-kB activation over unstimulated cells. Error bars depict SEM.

Given the previously reported capacity of MDP(d-Val¹) (compound 14) to act as an adjuvant (Table 1) and its ablated ability to induce Nod2 activation (Figure 2), we were interested in further testing whether this compound could decouple the inflammation-inducing capacity of MDP from the ability to induce adaptive immune responses. However, we observed that MDP(d-Val¹) was unable to stimulate KC or MIP-2 production from BMDM when compared to MDP in vitro (Figure 3A). Moreover, MDP(d-Val¹) that was injected i.p. did not induce an innate chemokine and cytokine response in vivo, as evidenced by the lack of KC and IL-6 induction in the serum at 2 h post-injection (Figure 3B). In contrast to previously reported results, we determined that MDP(d-Val¹) could not function as adjuvant as evidenced by the lack of detected OVA-specific serum IgG1 Abs after a prime-boost immunization regimen with OVA + MDP(d-Val¹) (Figure 3C). Together, our results indicate that MDP(d-Val¹) cannot induce innate and adaptive immune responses in vivo.

Figure 3.

In vitro and in vivo responses observed with MDP-derivative (d-Val¹). (A) Concentration in pg/ml of KC (left panel) and MIP2 (right panel) measured in the supernatants of BMDM stimulated with PBS, MDP or MDP(d-Val¹). Three replicates, one representative of two independent experiments. (B) Levels of KC (pg/ml) measured in the serum at 2 h post-challenge of C57Bl6 wild type (WT) or Nod2⁻^/⁻ mice injected with either PBS, 50 µg/mouse of MDP or 50 µg/mouse of MDP(d-Val¹) i.p. Minimum of three mice in each group, one representative of two independent experiments is shown. (C) Bar graph represents the concentrations of OVA-specific IgG1 in the serum of C57Bl6 WT or Nod2⁻^/⁻ mice 42 d after prime-boost (3–6 mice in each group) as measured by ELISA after initial immunization with 50 µg of OVA in sterile PBS, 50 µg OVA with 50 µg/mouse of MDP or 50 µg OVA with 50 µg/mouse of MDP(d-Val¹). Values depict the average of three replicates from two independent experiments. Error bars depict SEM. * P < 0.05.

As we observed that MDP(d-Glu²)-OCH₃ could activate Nod2 at lower concentrations than MDP in the luciferase assays, we were interested in further testing this compound in more physiological conditions.²⁶ In agreement with the results from the luciferase reporter assays, MDP(d-Glu²)-OCH₃ appeared to be a better inducer of KC and MIP-2 secretion than MDP when used to stimulate BMDM (Figure 4A). Moreover, MDP(d-Glu²)-OCH₃ at low concentrations (5 µg/mouse) induced higher levels of KC and IL-6 in the serum at 2 h post-injection in vivo compared with MDP at the same dose, reinforcing the idea that MDP(d-Glu²)-OCH₃ has a lower activation threshold than MDP (Figure 4B). At the higher dose of 50 µg/mouse, MDP(d-Glu²)-OCH₃ injection resulted in increased levels of KC and a trend for increased IL-6 in the serum at the same time point (Figure 4B). The increased potency of MDP(d-Glu²)-OCH₃ for inducing a rapid KC response was similar to that of N-glycolyl-MDP (Supplementary Figure 4), a compound that has previously been shown to exhibit increased Nod2-dependent innate immune responses over MDP.²⁶ Importantly, using Nod2 KO mice, we provide evidence that the host response to both MDP and MDP(d-Glu²)-OCH₃ was fully dependent on Nod2 (Figure 4B). Next, we determined that at the lower dose of 5 µg/mouse, MDP(D-Glu²)-OCH₃ exhibited a trend for more potent adjuvanticity compared to MDP for the generation of OVA-specific serum IgG1 Abs. In contrast, at the dose of 50 µg/mouse there was no observable difference in potency between MDP and MDP(d-Glu²)-OCH₃, as the immunization response likely reached a plateau. Finally, we found that MDP(D-Glu²)-OCH₃ was a much better agonist than MDP for activating human DC ex vivo as measured by the production of cytokine IL-6 and the chemokines MIP1α and MCP-1 (Figure 5), suggesting that this compound might be more sensitive for human cells compared to murine cells.

Figure 5.

MDP-derivative (d-Glu²)-OCH₃ induces enhanced cytokine and chemokine production by human dendritic cells compared to MDP. Bar graphs depict the levels of IL-6 (A), MIP1α (B) or MCP-1 (C) as measured by ELISA in the supernatants of purified human dendritic cells stimulated with 0.5, 5 and 50 µg/ml of MDP or MDP(d-Glu²)-OCH₃ for 16 h. Grouped data from one or two independent experiments, error bars depict SEM.

Figure 4.

In vitro and in vivo responses observed with MDP-derivative (d-Glu²)-OCH₃. (A) Concentration in pg/ml of KC (left panel) and MIP2 (right panel) measured in the supernatants of BMDM stimulated with PBS, MDP or MDP(d-Glu²)-OCH₃. Three replicates, one representative of two independent experiments. (B) Levels of KC and IL-6 (pg/ml) measured in the serum at 2 h post-challenge of C57Bl6 WT or Nod2^−/− mice injected with either PBS, 5 or 50 µg of MDP and 5 or 50 µg of MDP(d-Glu²)-OCH₃ i.p. Minimum of three mice in each group, one representative of two independent experiments is shown. (C) Bar graph represents the relative amounts of OVA-specific IgG1 in the serum of C57Bl6 WT mice 42 d after prime-boost (three mice in each group, one of two independent experiments) as measured by ELISA after initial immunization with 50 µg of OVA in sterile PBS, 50 µg OVA with 50 or 5 µg/mouse of MDP or 50 µg OVA with 50 or 5 µg/mouse of MDP(d-Glu²)-OCH₃. Error bars depict SEM. *P < 0.05.

Discussion

In this study, we found that many MDP-derivative compounds modified at the first or second amino acid can stimulate Nod2 equally well as MDP, while one in particular, MDP(d-Glu²)-OCH₃, was shown to be a more potent immune activator than MDP in the in vitro and in vivo stimulation assays we tested. In contrast, we also determined that MDP(d-Val¹), a compound that did not stimulate any Nod2-dependent NF-κB activation in vitro, was also unable to induce an inflammatory response in BMDMs or when injected into mice, and did not exhibit any capacity to function as an adjuvant in OVA-specific Ab responses.

We demonstrated that none of the MDP-derivatives that had the first amino acid of the d-configuration were capable of stimulating Nod2 in the luciferase assay and, given the results observed in vivo with MDP(d-Val¹) administration, we can speculate that no MDP-derivative that harbors a d-amino acid at the first position will stimulate either innate or adaptive immune responses. The discrepancies between our results and those obtained in the older studies could be owing to imprecision in the way the antigen-specific antibody response was measured previously. Based on our results, we speculate that an interesting avenue of investigation for future studies aiming at decoupling the inflammation and adjuvant effects of MDP would be to use MDP(l-Val¹) (compound 19) or MDP(l-Ser¹) (compound 16). Indeed, we found that these compounds still activated Nod2 (Figure 2), albeit less so than MDP, yet were still reported to exhibit adjuvanticity and a lack of anti-infectious properties (Table 1). It would be interesting to revisit whether MDP(l-Val¹) or MDP(l-Ser¹), which have higher thresholds for Nod2 activation, retain a significant ability to function as adjuvants. Indeed, a major issue that limits the use of Nod and TLR ligands as adjuvants in a clinical setting is the pyrogenic effects of these compounds. Therefore, identifying non-pyrogenic, or inflammation-decoupled, Nod2 ligands would represent a significant advance in the development of new adjuvants for clinical use.

Previously, it was reported that the conversion of the N-acetyl group on the MurNAc sugar to a N-glycolyl group, which occurs naturally in mycobacterial species by the enzyme N-acetylmuramic acid hydroxylase, results in a MP with a greater Nod2-stimulating activity.²⁶ In the present study, we have identified what appears to be the first chemical modification to the core amino acids of MDP that results in an analog with increased stimulatory capacity on Nod2. With this proof-of-principle observation, we can now speculate that the relatively modest increase in stimulatory capacity identified here (approximately fivefold) provided by the replacement of the terminal amide of MDP by a methyl ester could be optimized rationally by targeting the α-carboxyl group of d-Glu, and larger substitutions, such as ethyl, butyl or larger groups, would be worth testing. Moreover, it is noteworthy that such chemical modifications of the MDP core structure could be combined with the addition of acyl chains in order to generate MP derivatives that would be both more active and more bioavailable than MDP.

Many of the mutations in Nod2 that have been associated with increased risk for developing Crohn’s disease (CD) result in Nod2 proteins that are either completely unresponsive to MDP, such as the 3020insC frameshift mutant,^27–29 or have reduced signaling capacity, such as the newly identified S431L and N852S variants.³⁰ Therefore, it would be interesting to determine if administration of MDP analogs that exhibit increased Nod2 stimulatory activity, such as MDP(d-Glu²)-OCH₃ identified in this study, could restore normal levels of NF-κB activation in cells harboring certain CD-associated Nod2 mutations. Such a strategy would provide the basis for a targeted therapy, using MDP(d-Glu²)-OCH₃ or other second generation MP derivatives related to MDP(d-Glu²)-OCH₃, for CD patients with specific Nod2 mutations.

Footnotes

Funding

S.E.G. and D.J.P. are funded by grants from the Canadian Institute of Health Research (CIHR) and the Crohn's and Colitis Foundation of Canada. S.J.R. is supported by Banting and Best Graduate Scholarship from CIHR. J.G.M. is supported by a fellowship from the French Foundation for Medical Research.

Acknowledgements

We would like to thank Armelle Bohineust for her help with the human DC experiments.

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Supplementary Material

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Identification of a synthetic muramyl peptide derivative with enhanced Nod2 stimulatory capacity

Abstract

Keywords

Introduction

Materials and methods

Reagents

Luciferase assays

Mice

In vivo cytokine response

OVA-specific Ab response

Bone marrow-derived macrophage preparation

Isolation of human dendritic cells

Statistical analyses

Results

Discussion

Footnotes

Funding

Acknowledgements

References

Supplementary Material