Induction of Mincle by Helicobacter pylori and consequent anti-inflammatory signaling denote a bacterial survival strategy

Evasion of innate immune recognition is one of the key strategies for persistence of Helicobacter pylori, by virtue of its ability to modulate or escape the host innate immune receptors and signaling pathways. C-type lectin receptors (CLRs) predominantly expressed by macrophages are pivotal in tailoring immune response against pathogens. The recognition of glyco or carbohydrate moieties by Mincle (Macrophage inducible C-type lectin) is emerging as a crucial element in anti-fungal and anti-mycobacterial immunity. Herein, we demonstrate the role of Mincle in modulation of innate immune response against H. pylori infection. Our results revealed an upregulated expression of Mincle which was independent of direct host cell contact. Upon computational modelling, Mincle was observed to interact with the Lewis antigens of H. pylori LPS and possibly activating an anti-inflammatory cytokine production, thereby maintaining a balance between pro- and anti-inflammatory cytokine production. Furthermore, siRNA mediated knockdown of Mincle in human macrophages resulted in up regulation of pro-inflammatory cytokines and consequent down regulation of anti-inflammatory cytokines. Collectively, our study demonstrates a novel mechanism employed by H. pylori to escape clearance by exploiting functional plasticity of Mincle to strike a balance between pro-and anti-inflammatory responses ensuring its persistence in the host.


Results
Mincle expression elevated in H. pylori infected macrophages. Activation of Mincle has been studied in response to M. tuberculosis and various fungal species 31,32 . We investigated whether Mincle expression was upregulated upon H. pylori infection as well. Mincle mRNA expression was quantified in THP-1 cell line in response to H. pylori infection in vitro by using qRT-PCR and was validated by melting curve. A time course analysis of Mincle expression levels were carried out from 4 h to 24 h post infection and we observed that Mincle mRNA transcript levels were increased in a time dependent manner. Our results demonstrated a significant increase in the transcript level of Mincle mRNA as early as 6 h post infection and increased up to 200 fold at 24 h post infection as compared to uninfected THP-1 cells. (Fig. 1a). This suggests that THP-1 cells sense H. pylori and upregulate Mincle receptor possibly for initiating the immune response against H. pylori. To further investigate the surface expression of Mincle upon H. pylori infection on THP-1 cells, flow cytometry was performed and our results indicate enhanced levels of Mincle expression on the surface of infected cells as compared with the non-infected cells (Fig. 1b). This indicates that H. pylori strongly interacts with Mincle and its expression on macrophages increases after H. pylori infection.

Mincle expression is independent of live or heat killed H. pylori.
We also investigated the capability of heat killed and live H. pylori strains to induce Mincle expression in THP-1 cells. Our results demonstrated that the heat killed H. pylori also upregulated Mincle expression with no significant difference (Fig. 2). As Mincle possesses a carbohydrate recognizing domain (CRD) in its extracellular region, it may recognize any specific carbohydrate structure of H. pylori. Above results also hint at the possibility that Mincle could be interacting with any non-protein entity of H. pylori.
Mincle expression and cytokine secretion in THP-1 cells is independent of host cell contact. Further, we examined whether Mincle expression was host cell contact dependent or otherwise ( Fig. 3a). qRT-PCR analysis revealed that there was no significant difference in Mincle mRNA expression levels in H. pylori separated THP-1 cells and cells directly in contact with H. pylori (Fig. 3b). In our observation, Mincle was expressed in a host cell contact independent manner. We also investigated if the expression of pro and anti-inflammatory cytokines might also be regulated at the level of host cell contact/ or independent manner during infection of THP-1 cells. To investigate this, a similar strategy was employed and the level of TNF-α and IL-10 production was estimated both at transcript and protein levels. Interestingly, no significant difference in the production of TNF-α and IL-10 was observed for Scientific RepoRts | 5:15049 | DOi: 10.1038/srep15049 both directly infected cells as well as in separated THP-1 cells (Fig. 3c). One possible explanation for such an observation could be due to the fact that most of the Gram negative bacteria continuously release LPS during growth or in stationary phase 38 that might cross through the filters to induce expression of Mincle and inflammatory responses against H. pylori.

H. pylori shed LPS during infection with THP-1 cells. We next investigated if LPS shed by
H. pylori could pass through permeable filter supports, and were responsible for Mincle expression. Cell culture supernatants were collected from both directly and indirectly infected THP-1 cells from 12 h and 24 h time points and the presence of LPS was determined. We could observe a positive signal for LPS in the culture supernatant of indirectly infected macrophages as compared to the non-infected macrophages for both the time points (Fig. 4)  Expression of Mincle was normalized with PPIB (peptidlypropylisomerase B) expression and is presented relative to expression in untreated THP-1 cells. Values are mean ± s.e.m. The data were analysed by 1 way ANOVA followed by Tukey's multiple comparison test set as *P < 0.05, **P < 0.01, ***P < 0.001. (b) Surface expression of Mincle was analysed by flow cytometry in THP-1 cells post infection with H. pylori P12 and 26695 at 24 h. Infected THP-1 cells were stained with monoclonal anti-mincle antibody for Mincle expression and compared against un-infected cells (UI). Isotype antibody staining is represented in blue outline and uninfected/uninduced cells in red outline. GlcNAcb133Galb134Glc)] which are present in H. pylori LPS could accommodate in the ligand binding pocket of Mincle without steric clashes. Multiple sequence alignments of Mincle, DC-SIGN and DC-SIGNR revealed that residues recognizing saccharide moiety are invariant in SIGN and SIGN-R proteins whereas they were significantly different in the case of Mincle. Three polar or hydrophobic residues observed in DC-SIGN and DC-SIGNR were substituted to negatively charged residues in Mincle (Fig. 5a). Therefore, surface groove of Mincle that binds to ligand has negative potential. However, residues involved in coordination of Ca 2+ were conserved among these proteins. Based on this, we speculate that Mincle might have evolved to recognize different saccharides or glycolipids or Lewis antigens as outlined in Fig. 5b-f.
Though tri-saccharides (Gal-Fuc-Nac) given in Fig. 5b and c have different stereochemistry, they fitted well in the ligand binding surface of the Mincle. As predicted, 3 and 4 hydroxyl groups of galactose (Gal1) of the saccharide can coordinate the Ca 2+ of the protein molecule in case of both the tri-saccharides. This primary Ca 2+ coordinated interaction may lead to alignment of saccharide molecule on the groove of Mincle. Hydrophobic side chain of the V195 interacts with apolar face of the galactose in both ligands (5B and 5C), whereas, N-acetyl glucosamine (NAG or GlcNAc) was recognized differently by the Mincle due to the structural differences in tri-saccharides. Tri-saccharide shown in Fig. 5b, a polar surface of the NAG has an alignment with the aromatic side chain of Y201 with hydrophobic interactions. In contrast, NAG in Fig. 5c has hydrophobic contacts with aliphatic region of the E136 side chain. In both (Fig. 5b,c) the cases, variant R183 has hydrogen bond interactions with the hydroxyl group of sugar moiety. Similarly, variant E136 forms hydrogen bond interaction with the hydroxyl group of the sugar as shown in Fig. 5b,c.
In case of tetra-saccharide (Fuc-Gal-Fuc-NAG) shown in Fig. 5d, saccharide can align on the groove of the binding surface only when 3 rd and 4 th hydroxyl groups of Gal2 coordinate with the Ca 2+ . Side chain of V195 has hydrophobic interactions with the Gal2 whereas L199 could interact with fucose (Fuc3) and NAG4 monomers. Side chains of R183, E136 and Y201 residues form intermolecular hydrogen bond or effect polar interactions with the hydroxyl group of saccharides.
Comparison of ligand recognition surface of DC-SIGNR with Mincle revealed that Mincle has lengthier surface groove. Therefore, Mincle could recognize the longer oligosaccharides such as penta-and hexa-saccharides. We docked both penta (Gal-Fuc-NAG-Gal-Glu) and hexa (Gal-Fuc-NAG-Gal-Fuc-NAG)saccharides on the surface groove of Mincle and both fit well in the binding surface. In both the cases, Gal1 of the ligand is recognized through Ca 2+ mediated coordination. Side chains of residues on the surface involved in both hydrophobic and hydrogen bond interactions with the ligand. Terminal glucose (Glu) and NAG of penta-and hexa-saccharides are recognized by the main chain groups of S186. Recognition of first and terminal residues of ligand could help in in-line alignment of the saccharide on the ligand binding groove that could reinforce the network of secondary interactions between internal sugars and protein molecule. Our results showed that, binding of H. pylori LPS to Mincle might be mediated by fucose containing Lewis antigens. Comparative structural analyses demonstrated that Mincle's sugar binding surface is significantly different from DC-SIGNR and thus could recognize diverse linear saccharides.

Mincle drives anti-inflammatory responses upon H. pylori infection.
To further examine the functional significance of the increased levels of Mincle mRNA in infected THP-1 cells, we used siRNA mediated gene silencing approach to knockdown the Mincle gene in THP-1 cells and confirmed the same by qRT-PCR. The expression of Mincle mRNA was down regulated by 80% in Mincle silenced THP-1 cells (Fig. 6a). We also determined the role of Mincle receptors in H. pylori mediated innate immune functions. Interestingly, our results demonstrated a significant upregulation and secretion of TNF-α in Mincle silenced (knockdown) THP-1 cells as compared to the wild type THP-1 cells. The amount of IL-10 expressed and secreted by Mincle silenced THP-1 cells was also evaluated in comparison to the THP-1 wild type cells. Our results show that IL-10 induction was decreased significantly in Mincle silenced THP-1 cells relative to the wild type THP-1 cells (Fig. 6b). Collectively, our results suggest that Mincle expression occurs as an innate immune response towards H. pylori infection and it likely plays a crucial role in homeostasis of cytokine mediated pro and anti-inflammatory responses.

Discussion
Over the past few years, many studies described C-type lectins (CLRs) as primary mediators of diverse immune interactions, most notably in the recognition of various pathogens and host antigenic determinants 39 . However, their role in H. pylori infection has not been completely known. DC-SIGN is one of the CLRs whose activation has shown to be crucial for H. pylori infection 40 . Binding of H. pylori to DC-SIGN blocks maturation of naive T cells to Th1 cells; this prevents H. pylori clearance by host immune system 40 . Gringhuis et al. have also reported that DC-SIGN interacts with Lewis antigens, and modulates the cytokine expression 40 . With this background, we attempted to identify additional CLRs linked to H. pylori. The present study successfully deciphered the role of yet another CLR, Mincle, in mediating innate responses during H. pylori infection. The CLRs are made of transmembrane proteins with a characteristic carbohydrate recognition domain (CRD) 41 composed of two protein loops and two anti-parallel beta-sheets, made stable by highly conserved disulfide bonds plus four calcium binding sites. Owing to this arrangement, binding of ligands by CLRs is mostly a calcium dependent process 41,42 . The cytoplasmic domains of CLRs are frequently characterized as immune-receptor tyrosine-based activation motif (ITAM)-bearing adaptors such as Fc-receptor common γ chain (FcRγ ) 43 . Some C-type lectin receptors such as Dectin-1 and Mincle directly recognize beta glucans on the surfaces of fungi and mycobacterial glycolipid trehalose dimycolate (TDM), respectively 32 . The expression of Mincle gets triggered upon the onset of Mycobacterium tuberculosis, Streptococcus pneumoniae, Candidia albicans and Malassezia infections 42,44,45 ; this corroborates with our observations wherein Mincle expression was upregulated upon H. pylori infection (Fig. 1).
Our study revealed that Mincle mRNA transcript levels and surface expression of Mincle on PMA differentiated THP-1 cells were potentially triggered upon encounter with H. pylori in a time dependent manner. This strongly suggests an active involvement of Mincle in recognizing H. pylori during the course of infection. This is perhaps the first effort to elucidate the role of Mincle in H. pylori associated inflammation. We also found that the heat killed H. pylori retained stimulatory capability and was recognized by Mincle, ruling out the possibility of the involvement of any intact protein in recognition of H. pylori. Similar findings have also been reported by Yamasaki et al. wherein they demonstrated the ability of heat killed pathogenic fungus Malassezia to induce NFAT-GFP activation in Mincle reporter cells 31 . Considering the ability of both the live and heat killed H. pylori to induce Mincle, it is interesting to ascertain if the enhanced expression of Mincle was dependent purely on the interaction or any direct/indirect effect of host cell contact. In this context, our data indicated that Mincle expression was  independent of host cell contact and there was no significant difference in Mincle expression either in directly or in indirectly infected THP-1 cells (Fig. 3). One possible explanation for this observation could be due to gastric epithelial cells secreting IL-8 when in contact with H. pylori, to recruit monocytes and macrophages to the gastric mucosa 33 . The peripheral inflammatory environment during chronic H. pylori infection might be dominated by macrophages where H. pylori LPS interacts with Mincle. Further, mononuclear cell infiltration in the lamina propria is seen as a cardinal feature of H. pylori induced chronic infection which has also been demonstrated at the level of gastric tissue samples of infected patients 46,47 . Similarly, we observed the expression of TNF-α and IL-10 by THP-1 cells in a contact independent manner (Fig. 3c). Our observations also confirm that the production of pro-and anti-inflammatory cytokines is not regulated at the host cell contact and it is more of a generic response towards the pathogen. Indeed, LPS is known to be a common trigger of innate immune responses 48 , and it is shown to be released by different Gram negative bacteria during both in vitro and in vivo growth 38,49 .
We also observed similar results wherein H. pylori releases LPS during infection that could pass through the permeable filter supports as detected by the LAL test (Fig. 4). Our results showed that the LPS released from H. pylori increases in a time dependent manner, which is essentially in agreement with the observation that Mincle expression also increased over time. Given these observations, it is clear that release of the LPS during H. pylori infection might be responsible for Mincle activation and consequent pro-and anti-inflammatory cytokine production. Ever since the discovery of Lewis antigens in the LPS of H. pylori, a number of different biological functions such as increase in colonization, avoidance of host recognition, immune cells modulation and triggering of gastric autoimmunity have been associated with Lewis antigens 20,50 . Various Lewis antigens such as Lewis a , Lewis b , Lewis x and Lewis y are known ligands for DC-SIGN 40,51,52 . Moreover, dendritic cells and macrophages are the main targets for LPS 53 . Therefore, it is possible to surmise that the Lewis antigens might also mediate LPS binding to Mincle. Our hypothesis of LPS binding to Mincle through Lewis antigens is supported by the docking studies (Fig. 5). All the Lewis antigens outlined in Fig. 5 were fitted well with optimum network of interactions and minimum steric clashes and that indicated the possible interaction of Lewis antigens of H. pylori LPS with Mincle. Our observations in this context supported the interaction of Lewis x , Lewis a , Lewis b and human CD15 with Mincle.
We also found that Mincle silenced THP-1 cells enhanced production of pro-inflammatory cytokine, TNF-α and abrogated the production of anti-inflammatory cytokine IL-10 when compared with wild type macrophages. Recently, it has been reported that DC-SIGN which recognized the fucose ligands of H. pylori could also down regulate the pro-inflammatory signaling pathway 40 . Conversely, our results do not align with the observations reported with pathogens such as Mycobacterium and HIV which express mannosylated ligands and upregulate pro-inflammatory signaling pathways 54,55 . It has been demonstrated that Mincle induction on macrophages in response to BCG increases the production of TNF-α and MIP-2 in vitro 55 . This disparity could be due to a very different composition of capsular antigens in H. pylori.
Given that most of the virulence genes of H. pylori encode pro-inflammatory functions, upregulation of Mincle seems to be one of the possible mechanism by which H. pylori obliterates excessive pro-inflammatory cytokine secretion by IL-10 induction and may dampen inflammation in its own favor. In view of this possibility, our study suggests that other than the TLRs, Mincle induced anti-inflammatory cytokine production may contribute to maintenance of chronic persistence of H. pylori. It also provides an insight on differential role of fucosylated and mannosylated Mincle ligands in altering innate immunity to specific pathogens. However, to further decipher the role of Mincle in host defense or H. pylori survival, in-vivo mouse models need to be established.
In conclusion, we propose an interaction of H. pylori LPS and its released form with Mincle. We earlier hypothesized that during infection, shed LPS cross the epithelial lining and reach lamina propria as well as the endothelial layer of inflamed gastric mucosa and consequently increase the secretion of IL-6, IL-8 and IL-1β , which in turn attract the monocytes and macrophages to the site of infection. At this stage, Mincle might interact with Lewis antigens of LPS and lead to the production of IL-10. Therefore, upregulation of Mincle by LPS of H. pylori possibly fine tunes adaptation of H. pylori strains to their individual hosts; this might facilitate avoidance of detrimental host responses thus contributing crucially during chronic H. pylori infection.
Given that H. pylori eradication has become uncertain due to emergence of increased antimicrobial resistance 56 , the development of newer interventions such as immunotherapeutics and vaccines has become inevitable; this requires a comprehensive understanding of host-pathogen interactions. A systematic unraveling of host mediators of H. pylori-induced pathogenesis could possibly identify potential drug targets for therapeutic intervention against H. pylori-associated disease. With the understanding of the innate immune modulation triggered by H. pylori and the interplay of pro-inflammatory and anti-inflammatory signals, the immune response could be fine-tuned therapeutically to successfully eradicate the bacterium. Our observations therefore constitute important co-ordinates of innate immune functions mediated by H. pylori and therefore could be helpful in strengthening immune based control and eradication strategies. Estimation and analysis of Mincle expression: RNA isolation, qRT-PCR and Flow cytometry. Total RNA was extracted by using Trizol (sigma), and 1 μ g of purified RNA was treated with DNase (sigma) and reverse transcribed by using first strand synthesis system (Invitrogen Life technologies) as per the manufacturer's instructions. qRT PCR was carried out by using Eppendorf real time machine by utilizing the primers as given in Table 1. Briefly, the reaction was performed in 10 μ l volume containing 5 μ l of SYBR green (Bioline), 0.2 μ l forward primer and 0.2 μ l reverse primer, 40 ng c-DNA and remaining amount of DNase-RNase free water (Invitrogen, Life technologies). Real time PCR was performed as follows for all Mincle, TNF-α and PPIB 95 °C for 10 min, 95 °C for 15 sec, 58 °C for 15 sec, and 72 °C for 15 sec. Mean fold changes were analyzed by ∆∆CT method as described earlier 57 . To determine the surface expression of mincle, PMA differentiated THP-1 macrophages were incubated with 10 μ g/ml mouse anti-mincle mAb or isotype matched control IgG2b antibody for 60 min at 4 °C followed by incubation with FITC-conjugated goat anti mouse IgG (sigma) for another 45 min at 4 °C . Cells were washed and resuspended in 1X PBS with 1% BSA. The fluorescence was measured by BD-FACS Canto II and results were analyzed by Flowjo software.

Computational modeling of Mincle interaction with Lewis antigens. Oligosaccharides used for
docking were modeled and energy minimized using, GLYCAM, a web based server (http://glycam.org/). DC-SIGNR (PDB ID: 1K9J) and DC-SIGN (PDB ID: 1K9I) complexed with GlcNAc2Man are used as template for manual docking of different oligosaccharides on Mincle. Ligand free structure of Mincle is superposed on DC-SIGNR and DC-SIGN using the COOT program 58 GlcNAc2Man saccharide, present in the complex structures, was used as reference for determining the orientation of different oligosaccharides outlined in the Fig. 5B-F. Following criteria were used in manual docking of the oligosaccharide: OH groups of sugar of one of the monomers should be positioned in such an orientation that could able to coordinate with Ca 2+ . Ca 2+ coordination with OH groups should lead to an in-line alignment of sugar moieties on the surface groove of the Mincle. Alignment of saccharide should have minimum short contacts and optimum interactions with residues of the protein molecule. We carried out validation for

S. No
Gene name Primer sequences Reference

Lipopolysaccharide (LPS) estimation. Release of H. pylori LPS during direct and indirect infection
in THP-1 cells was measured in culture supernatant at different time intervals. The level of shed LPS was determined by Limulus amebocyte lysate assay (LAL) kit (Pierce Themo Scientific) according to the manufacture's instruction. 50 μ l of cell culture supernatant was added in triplicate to 50 μ l of LAL in a pyrogen-free microtiter plate. The mixture was incubated at 37 °C for 10 min, and 100μ lof chromogenic substrate solution was added and color development was terminated by addition of 20% acetic acid. The optical density was measured at 410 nm.
Statistical Analysis. Statistical calculations were performed by using GraphPad Prism 5 software. For ELISA and qRT PCR, statistical evaluation was performed by using student's t-test and one way ANOVA and p ≥ 0.05 was considered non-significant.