Nod1 Imprints Inflammatory and Carcinogenic Responses Toward the Gastric

Author Manuscript Published OnlineFirst on January 29, 2019; DOI: 10.1158/0008-5472.CAN-18-2651 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Nod1 imprints inflammatory and carcinogenic responses toward the gastric

pathogen Helicobacter pylori

Key Words: H. pylori, Nod1, gastric cancer

Running Title: Nod1 suppresses H. pylori-induced gastritis and gastric cancer in 2

mouse models of infection

Giovanni Suarez1, Judith Romero-Gallo1, Maria B. Piazuelo1, Johanna C. Sierra1,

Alberto G. Delgado1, M. Kay Washington2, Shailja C. Shah1, Keith T. Wilson1,2, and

Richard M. Peek, Jr.1,2

Departments of 1Medicine and 2Pathology, Microbiology, and Immunology;

Vanderbilt University Medical Center, Nashville, TN

Correspondence: Richard Peek, 2215 Garland Avenue, Nashville, TN, 37232-2279

Phone: 615-322-5200; Fax: 615-343-6229; E-mail: [email protected]

Conflicts of Interest: The authors declare no potential conflicts of interest

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 29, 2019; DOI: 10.1158/0008-5472.CAN-18-2651 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract: Helicobacter pylori is the strongest known risk for gastric cancer. The H.

pylori cag type IV secretion system is an oncogenic locus which translocates

peptidoglycan into host cells where it is recognized by NOD1, an innate immune

receptor. Beyond this, the role of NOD1 in H. pylori-induced cancer remains

undefined. To address this knowledge gap, we infected two genetic models of Nod1

deficiency with the H. pylori cag+ strain PMSS1: C57BL/6 mice which rarely develop

cancer, and INS-GAS FVB/N mice which commonly develop cancer. Infected

C57BL/6Nod1-/- and INS-GASNod1-/- mice acutely developed more severe gastritis, and

INS-GASNod1-/- mice developed gastric dysplasia more frequently compared to

Nod1+/+ mice. Because Nod1 genotype status did not alter microbial phenotypes of

in vivo-adapted H. pylori, we investigated host immunological responses. H. pylori

infection of Nod1-/- mice led to significantly increased gastric mucosal levels of Th1,

Th17, and Th2 cytokines compared to Nod1 wild-type mice. To define the role of

specific innate immune cells, we quantified cytokine secretion from H. pylori-infected

primary gastric organoids generated from WT or Nod1-/- mice, that were co-cultured

with or without WT or Nod1-/- macrophages. Infection increased cytokine production

from gastric epithelial cells and macrophages and elevations were significantly

increased with Nod1 deficiency. Further, H. pylori infection altered the polarization

status of Nod1-/- macrophages compared to Nod1+/+ macrophages. Collectively,

these studies demonstrate that loss of Nod1 augments inflammatory and injury

responses to H. pylori. Nod1 may exert its restrictive role by altering macrophage

polarization, leading to immune evasion and microbial persistence. Statement of

Significance: Findings suggest that manipulation of NOD1 may represent a

novel strategy to prevent or treat pathologic outcomes induced by H. pylori

infection.

Introduction Helicobacter pylori is the most common bacterial infection worldwide, colonizing

more than 4.4 billion persons (1). Infection with this pathogen also represents the

strongest known risk factor for gastric adenocarcinoma, the third leading cause of

cancer-related death (2). However, only a percentage of colonized persons ever

develop gastric neoplasia (3). One strain-specific virulence locus that augments

cancer risk is the cag pathogenicity island (PAI), which encodes a type IV secretion

system (T4SS) that translocates the oncoprotein CagA and microbial DNA into

gastric epithelial cells (4-6). Following T4SS-mediated delivery, intracellular CagA

undergoes tyrosine phosphorylation (5), and activates a eukaryotic phosphatase

(SHP-2), leading to carcinogenic cellular responses.

In addition to CagA and DNA, the cag T4SS delivers peptidoglycan into host cells (7-

11). Peptidoglycan is also delivered intracellularly via outer membrane vesicles (12).

NOD1, which is expressed by most gastrointestinal epithelial cells, is an innate

immune receptor and intracytoplasmic sensor of peptidoglycan components. Most

gastrointestinal epithelial cells express NOD1 and activation of NOD1 by the

muropeptide -D-glutamyl-meso-diaminopimelic acid (iE-DAP) leads to NF-B-

dependent cytokine production as well as induction of autophagy (13, 14). NOD1 is

also expressed and activated within macrophages in vivo (15-17). NOD1 sensing of

H. pylori peptidoglycan induces NF-B activation and expression of type I IFN via

IFN-Regulatory-Factor 7, MIP-2, and -defensin (7, 8, 13, 18). In humans, genetic

variation in ATG16L1, which encodes a key effector of NOD1-dependent autophagy

and inflammation, alters susceptibility to H. pylori infection (19).

However, NOD1 activation is tightly regulated by a negative autocrine feedback

system, in which NOD1-dependent effectors such as AP-1 and TRAF3

concomitantly suppress the downstream effects of NOD1 activation (18, 20-22). We

previously demonstrated that H. pylori-induced injury can be significantly attenuated

in vitro and in outbred Mongolian gerbils by pre-activation of NOD1 (22), yet the role

of aberrant NOD1 activation by H. pylori in gastric carcinogenesis has not been fully

investigated. Therefore, we utilized 2 mouse models of gastric injury and cancer on

different genetic backgrounds to precisely define the role of Nod1 in inflammation

and inflammation-related cancer that develops in response to H. pylori.

Methods

Bacteria

H. pylori strains PMSS1 (23) and 7.13 (24), both cag+ strains, were maintained on

TSA blood agar plates (25). For in vitro and in vivo experiments, H. pylori was

cultured in Brucella broth (Becton Dickinson) supplemented with 10% heat-

o inactivated new calf serum (Atlanta Biologicals) overnight at 37 C and 5% CO2.

Cells

AGS cells (ATCC CRL-1739) were purchased from ATCC, tested for mycoplasma

contamination on July 10, 2018, and determined to be mycoplasma free. Cells

passed for <10 passages were grown in RPMI-1640 media (Gibco) supplemented

with 10% heat-inactivated fetal bovine serum (FBS) (Atlanta Biologicals) at 37oC and

5% CO2. L-WRN fibroblasts (ATCC CRL-3276) were obtained from ATCC, tested for

mycoplasma contamination on July 10, 2018, and determined to be mycoplasma

free. L-WRN cells passed for <10 passages were grown in Advanced DMEM media

(Gibco) supplemented with 10% FBS, 500 g/mL G418 (Gibco), 500 g/mL

o hygromycin (Invitrogen) at 37 C and 5% CO2. Once cells became confluent,

antibiotics were removed, and supernatants were collected. Mouse primary gastric

epithelial cell monolayers were generated as reported (26). Briefly, gastric glands

harvested from WT or Nod1-/- mice were embedded into Matrigel (Corning) and

o cultured in 50% L-WRN conditioned media at 37 C and 5% CO2. Once glands

formed 3D gastroids, they were trypsinized, and plated in collagen-coated plates or

o transwell filters (Corning) in 5% L-WRN conditioned media at 37 C and 5% CO2 to

convert to 2D monolayers. Cell monolayers were then infected with H. pylori at a

multiplicity of infection (MOI) of 30.

Bone marrow-derived macrophages were obtained from femurs of C57BL/6 WT and

Nod1-/- mice. Briefly, marrows were treated with red blood cell lysis buffer (Becton

Dickinson) and washed with PBS, and recovered white blood cells were plated in

DMEM media (Gibco) supplemented with 10% FBS and 20 ng/mL of M-CSF

o (Peprotech) for 6 days at 37 C and 5% CO2 for differentiation.

Animals

All procedures were approved by the Animal Care Committee of Vanderbilt

University. All mouse strains were bred and maintained in the same animal facility.

C57BL/6 Nod1-/- mice were kindly provided by Dr. Dana Philpott from the University

of Toronto. FVB/N INS-GAS Nod1-/- mice were generated by crossing FVB/N INS-

GAS Nod1+/+ mice with C57BL/6 Nod1-/- mice for 12 generations. Nod1-/- and INS-

GAS+/+ genotypes were confirmed by PCR and qPCR respectively. Mice were

housed in the Animal Care Facility of Vanderbilt University Medical Center in

standard plastic cages in a room with a 12-hour light/dark cycle at 21o to 22oC and

fed a standard rodent chow (Purina, 5L0D). Access to food and water was free

throughout all experiments. No special pretreatments (acid inhibition, antibiotics)

were used prior to orogastric H. pylori inoculation or before sacrificing the animals.

For C57BL/6 mice, both females and males were used; for FVB/N INS-GAS Nod1+/+

and Nod1-/- mice, only males were used in this study (Supplemental Table 1). Mice

6-8 weeks of age were challenged with 1x109 H. pylori at 2 time-points (days 0 and

2) as previously described (27) except for the 2 day time-point, where animals were

given a single challenge. All mice appeared healthy with no signs of distress noted 6

throughout the infection period up until the time of sacrifice. Serum samples and

gastric tissue were harvested. A single pathologist scored indices of inflammation,

injury, and cancer as described previously (28). Specifically, the following variables

were graded on a 0 to 3 scale (0, none; 1, mild; 2, moderate; 3, severe) in the gastric

antrum and body: acute inflammation (polymorphonuclear cell infiltration) and

chronic inflammation (mononuclear cell infiltration independent of lymphoid follicles);

thus a maximum inflammation score of 12 was possible for each animal. Dysplastic

mucosa was graded as 0 (absent), 1 (focal), or 2 (extensive) and consisted of

irregular, angulated, and occasionally cystically dilated glands with enlarged

overlapping hyperchromatic nuclei. Carcinoma was defined as irregular, angulated,

cystically dilated glands with occasional cribriform architecture in the submucosa

and muscularis propria, spreading laterally to the surface mucosal component (28).

For quantitative H. pylori culture, serial dilutions of homogenized tissue were plated

on selective antibiotic TSA-blood agar plates (27).

Multiplex bead array

Gastric linear strips extending from the squamocolumnar junction through the

proximal duodenum were lysed in 200 L of IP lysis buffer (Thermo Scientific)

containing protease and phosphatase inhibitors (Roche). Lysates were diluted 1:3 in

assay buffer and mixed with magnetic beads according to the manufacturer’s

instructions (Millipore) (27). Data were acquired and analyzed using the Millipore

software platform.

RT2 profiler PCR array

RNA from primary gastric epithelial cell monolayers was isolated using the RNeasy

kit (Qiagen). Total RNA was treated with RNase free-DNase (Promega) overnight

and then used for cDNA synthesis (Applied Biosystems). Real time PCR master mix,

plates and running protocols were performed following the manufacturer’s

instructions for the mouse NF-B gene target array (Qiagen). Data were analyzed

using the online Data Analysis Center provided by Qiagen.

Real time RT-PCR

Total RNA isolated from bone marrow-derived macrophages or AGS gastric

epithelial cells (CRL-1739) was subjected to overnight treatment with RNase-free

DNase, and then reverse transcribed to cDNA. Quantitative PCRs were performed

to determine relative differences in expression levels of Nos2, TNF, IL-10, TGF,

Light, and Ym1, in murine macrophages and CXCL8 and CXCL2 in AGS cells.

Results were then normalized to corresponding levels of GAPDH. For NOD1

inhibition studies, AGS cells were transfected with a mix of shRNAs targeting NOD1

as previously described (22). Colonies were selected using puromycin (10 μg/mL)

and tested for NOD1 expression by real-time RT-PCR and Western blot.

CagA translocation

AGS cells co-cultured with H. pylori were lysed in RIPA buffer containing

phosphatase and protease inhibitors. Proteins were separated using 6% SDS-PAGE

mini gels, transferred to PVDF membranes (Thermo Scientific) and membranes

were blocked with 1% BSA (Sigma) overnight. Incubation with primary antibodies

(mouse anti-PY99 (Santa Cruz Biotechnology), rabbit anti-CagA (Austral Biologicals),

and mouse anti-GAPDH (Millipore) was performed for 1 hour followed by addition of

respective HRP-conjugated secondary antibodies (anti-mouse-HRP or anti-rabbit-

HRP; Promega). The reaction was developed using ECL (Thermo Scientific).

c-Jun immunofluorescence staining

Monolayers of primary gastric epithelial cells derived from C57BL/6WT, C57BL/6Nod1-/-,

FVB/N INS-GASNod1+/+, and FVB/N INS-GASNod1-/- mice were infected for 1 hour with

H. pylori strains 7.13 or PMSS1. After infection, monolayers were subjected to c-Jun

immunofluorescence staining as previously described (26). Briefly, cells were fixed

with 4% paraformaldehyde (Thermo Scientific) for 1 hour and then blocked with 5%

goat serum (Sigma) in PBS for 1 hour. Samples were incubated with an anti-

phospho-c-Jun antibody (1:500 dilution; Cell Signaling) overnight at 4C. Samples

were then incubated with Alexa 488-anti-rabbit (1:1000; Invitrogen), Alexa 546-

Phalloidin (1:500; Invitrogen), and Hoechst 33342 (1:1000; Invitrogen) for 1 hour at

room temperature. Slides were mounted using ProLong Glass (Invitrogen), and

images were acquired in an Olympus FV-1000 confocal microscopy.

Statistical analysis

The Mann-Whitney test was used for 2 group comparisons, while one-way analysis

of variance with Newman-Keuls post-test was used for multiple group comparisons.

Data were plotted and analyzed using Prism V. 5b (GraphPad software Inc).

Statistical significance was set at a two-tailed p-value of <0.05.

Results Nod1 suppresses the early inflammatory response to H. pylori in C57BL/6 mice

We previously demonstrated that pre-activation of NOD1 leads to attenuated

cytokine production by H. pylori in vitro and reduced inflammation in an outbred

model of infection (Mongolian gerbils) (22). We also showed that expression of

NOD1 per se and its target genes were significantly decreased in human gastric

cancer specimens compared to samples with gastritis alone (22). Therefore, we

sought to define mechanisms that regulate this potential anti-inflammatory response

within the context of gastric carcinogenesis using genetic models of NOD1

deficiency.

C57BL/6WT and C57BL/6Nod1-/- mice were challenged with the mouse-adapted H.

pylori cag+ strain PMSS1 or broth alone, and stomachs were harvested and

analyzed at 2 (acute), 20 (subacute), and 90 (chronic) days post-challenge. All mice

challenged with H. pylori were successfully infected. H. pylori-infected C57BL/6Nod1-/-

mice developed significantly more severe gastric inflammation 20 days post-

challenge compared to their infected wild-type counterparts (p<0.05, Figure 1A, 1B).

However, by 90-days post-challenge, both groups had similarly elevated

inflammation scores (p=NS).

To define mechanisms underpinning these temporal differences in inflammation, we

initially investigated whether microbial factors contributed to these phenotypes. No

significant differences in levels of H. pylori colonization were present between WT

and Nod1-/- mice at any time-point (Figure 1A). We next determined whether

selective pressure exerted by the genetic loss of Nod1 affected H. pylori virulence

phenotypes in vivo. H. pylori strains were recovered from infected wild-type or Nod1-

/- mice and assessed for the ability to translocate CagA in vitro as a measure of cag

T4SS functionality. AGS gastric epithelial cells were infected with in vivo-adapted H.

pylori strains and the ratio of phosphorylated CagA (intracellular) to total CagA was

quantified by Western blot. The vast majority of strains harvested at 20 days post-

infection harbored a functional cag T4SS. However, as previously reported (25, 29),

loss of cag T4SS function was present in many of the in vivo-adapted isolates

harvested at 90 days post-challenge (Figure 1C). Since activation of NOD1 is cag-

dependent (7), this may partially explain the lack of difference in severity of

inflammation between infected WT or Nod1-/- mice at the 90 day time-point.

Importantly, host Nod1 status had no effect on T4SS function at either time-point

(Figure 1C). Thus, on a genetically defined background, loss of Nod1 resulted in

more severe subacute inflammation and injury within the context of H. pylori

infection and in conjunction with strains that harbored a functional cag T4SS;

however, Nod1 deficiency did not alter H. pylori cag T4SS phenotypes.

Loss of Nod1 alters cytokine production within H. pylori-infected gastric mucosa

Having shown that H. pylori increases inflammation in Nod1-/- mice despite similar

cag T4SS function (Figure 1), we next performed an unbiased survey to assess the

role of Nod1-mediated immune effectors that may contribute to differences in

inflammation within uninfected or infected gastric tissue. Infection with H. pylori 11

induced an increase in the levels of numerous chemokines, as well as Th1, Th2, and

Th17 cytokines in gastric mucosa harvested from both WT and Nod1 deficient mice

when compared to uninfected controls. For ease of presentation, we have included

cytokines and chemokines in Figure 2 that 1) were statistically different in terms of

levels of expression between Nod1+/+ and Nod1-/- mice and 2) could be classified as

either chemokines or Th1, Th2, or Th17 cytokines. The complete sets of data are

contained in Supplemental Tables 2-4. Expression levels of archetypal Th1 secreted

cytokines such as IL-12, Th1-associated pro-inflammatory chemokines such as IL-1,

the Th17 cytokines IL-17 and IL-23, and the Th2 cytokine IL-9 were further

increased in H. pylori-infected C57BL/6Nod1-/- compared to infected C57BL/6WT mice

at each time point (2, 20, 90 days post-challenge) (Figure 2A, 2B, 2C, Supplemental

Tables 2-4). In contrast to universal elevation of these cytokines and chemokines

throughout the duration of infection, there were also more selected differences

between H. pylori-infected WT versus Nod1 deficient mice that varied by time-point

(Figure 2A, 2B, 2C, Supplemental Tables 2-4). Specifically, at 20 days post-

challenge when inflammation was significantly increased in Nod1 deficient mice

(Figure 2B), levels of G-CSF, IL-7, IP-10, IFN-, IFN- and IL-4 were exclusively

increased in infected C57BL/6Nod1-/- compared to C57BL/6WT mice. In addition, the

overall number of cytokines and chemokines that were elevated in infected WT mice

increased from 2 to 90 days post-challenge (Figure 2A, 2B, 2C). Thus, in addition to

altering the level of inflammation in a time-dependent manner, loss of Nod1 also

temporally modified the portfolio of chemokine and cytokine expression within H.

pylori-infected gastric mucosa.

H. pylori differentially alters cytokine production in primary gastric organoid systems

in a Nod1-dependent manner

Our findings in gastric mucosa provided important insights into mechanisms through

which Nod1 may restrain early pro-inflammatory responses to H. pylori. However,

gastric tissue contains a myriad of cell types, including epithelial cells and

macrophages among others. Gastroids are polarized, replenishable epithelial culture

systems that can be readily generated from non-transformed gastric epithelium (30).

We have previously developed and optimized gastroid models of H. pylori infection

originating from murine tissue (31); therefore, we capitalized on this reductionist ex

vivo model to begin to dissect the role of specific cell types in regulating phenotypes

linked to Nod1 deficiency (Figure 3). Polarized 2-dimensional gastroid monolayers

were generated from uninfected C57BL/6WT and C57BL/6Nod1-/- mice, infected ex

vivo with H. pylori strains PMSS1 or 7.13 for 6 or 24 hours, and then co-culture total

mRNA was subjected to real-time RT-PCR to quantify expression levels of NF-B

target genes. Compared to expression levels in gastroids generated from wild-type

mice (Figure 3A, Supplemental Figure 1A), expression levels of numerous NF-B

target genes were significantly increased in infected Nod1 deficient gastroids at both

6 (Figure 3B, Supplemental Figure 1B) and 24 (Figure 3C, Supplemental Figure 1C)

hours post H. pylori challenge (Figure 3, Supplemental Figure 1, Supplemental

Tables 5-9). Of note, the magnitude of these effects were amplified following

infection with H. pylori strain PMSS1 versus strain 7.13, which likely reflects the

genetic diversity inherent within this pathogen. Specific targets up-regulated in Nod1

deficient gastroids infected by either H. pylori strain that were also up-regulated

acutely in vivo included Cxcl1 (e.g., KC). We therefore validated expression of KC at

the protein level in gastric epithelial monolayers by demonstrating that KC protein

was not only increased by H. pylori infection but that loss of Nod1 significantly

augmented this effect (Figure 3D). These data complement our previously published

results (22) demonstrating that suppression of Nod1 in gastric epithelial cells

augments H. pylori-induced NF-B activation. However, based on prior data

implicating MAP kinase activation as well as NF-B activation in mediating

downstream effects of Nod1 (32, 33), we also investigated the role of MAP kinase

activation within the context of Nod1 in our gastroid system. As shown in Figure 3E

and 3F, we co-cultured gastroids isolated from Nod1+/+ or Nod1-/- mice with H. pylori

and examined c-Jun activation by immunofluorescence. In contrast to the pattern

observed with NF-B, Nod1 deficiency suppressed c-Jun activation in response to H.

pylori infection (Figure 3E, 3F). Thus, Nod1 can exert differing effects on signaling

pathways following infection with H. pylori. Collectively, these results indicate that

gastric epithelial cells likely represent an important source of cytokine production

that is under Nod1-dependent control during H. pylori infection.

NOD1 regulates cytokine production in multiple innate immune cells following H.

pylori infection

Gastritis is an initiating event for the development of most gastric adenocarcinomas,

and macrophages are required for gastritis to develop in response to H. pylori (34).

Therefore, we enriched our gastroid monolayer system by adding macrophages

derived from WT or Nod1 deficient mice to epithelial cells derived from the same

mice.

Gastroid epithelial monolayers from uninfected WT or Nod1 deficient mice were

seeded in the upper chamber of a transwell system and macrophages from the

same mice were placed in the lower chamber in different combinations. H. pylori

strain PMSS1 was then added to epithelial monolayers for 24 hours and cytokine

production was quantified in supernatants. Loss of Nod1 in H. pylori-infected

epithelial cells co-cultured with WT macrophages resulted in significantly enhanced

levels of chemokines and cytokines including KC, MIP-2, IL-6, MCP-1 and IL-1

(Figure 4A) when compared to infected WT epithelial cells co-cultured with WT

macrophages. However, loss of Nod1 in both epithelial cells and macrophages

resulted in a markedly enhanced cytokine response to H. pylori (Figure 4B). These

results indicate that loss of Nod1 in multiple innate immune cells likely contributes to

enhanced inflammation observed in H. pylori-infected Nod1-deficient mice.

We also capitalized on this system to assess cytokine expression in macrophages

per se. RNA was isolated from macrophages subjected to uninfected and infected

transwell co-cultures and expression of prototype M1 (classically activated), M2

(alternatively activated), and Mreg (regulatory) macrophage genes was determined

by real-time RT-PCR. Uninfected WT macrophages harbored a predominant M2

phenotype, which shifted dramatically to an M1 phenotype after H. pylori infection. In

contrast, uninfected Nod1 deficient macrophages displayed a more profound M2

phenotype than uninfected WT macrophages; following H. pylori infection, the profile

of Nod1 deficient macrophages shifted to a hybrid M1/M2 phenotype. There were no

significant differences in Mreg profiles between macrophages from WT or Nod1 15

deficient mice (Figure 4C). Thus, loss of Nod1 alters cytokine profiles in both

epithelial cells and macrophages during co-incubation experiments in response to H.

pylori.

Loss of Nod1 accelerates gastric carcinogenesis in a mouse model of stomach

cancer

Our studies described above using a mouse model of H. pylori-induced gastritis

demonstrated that C57BL/6Nod1-/- mice develop more severe inflammation in

response to H. pylori infection than C57BL/6WT mice (Figure 1A, 1B). Since specific

genetic backgrounds can influence disease outcome in different mouse models (35),

we next determined whether the increased inflammatory phenotype induced by loss

of Nod1 in C57BL/6 mice could be recapitulated in H. pylori-infected mice on a

different genetic background that are also susceptible to gastric cancer.

INS-GASNod1+/+ and INS-GASNod1-/- mice on a FVB/N background were challenged

with H. pylori strain PMSS1 or broth alone, and stomachs were harvested and

analyzed 2, 20, 40, and 90 days post-challenge. The 40 day time-point was added

due to accelerated inflammation and damage previously observed in H. pylori-

infected INS-GAS mice at this time-point (36). All mice challenged with H. pylori

were successfully infected. H. pylori-infected INS-GASNod1-/- mice developed

significantly more severe acute and chronic inflammation compared to their infected

wild-type Nod1 counterparts at 20 and 40 days post-challenge (Figure 5A, 5B). Of

interest, loss of Nod1 per se in uninfected INS-GAS mice led to increased levels of

inflammation and injury at 40 and 90 days post-challenge compared to uninfected

INS-GAS mice with a wild-type Nod1 genotype (Figure 5A, 5B). Thus, on two

genetically distinct backgrounds, loss of Nod1 resulted in a time-dependent pattern

of more severe inflammation and injury within the context of H. pylori infection.

Unlike C57BL/6 mice, which do not develop cancer prior to 15 months post H. pylori

infection (37), INS-GAS mice rapidly develop pre-malignant lesions as early as 6-12

weeks following H. pylori challenge (36). At 40 days post-challenge, none of the

uninfected or H. pylori-infected INS-GASNod1+/+ developed gastric dysplasia. However,

loss of Nod1 led to the development of dysplasia in 50% of uninfected mice, and the

frequency of dysplasia rose significantly in conjunction with H. pylori infection (Figure

5C). At 90 days post-challenge, these effects were magnified. Infection of INS-

GASNod1+/+ with H. pylori significantly increased the prevalence of dysplasia

compared to uninfected WT controls, which did not demonstrate any evidence of

premalignant lesions. In uninfected INS-GASNod1-/- mice, the prevalence of gastric

dysplasia was significantly higher than in uninfected INS-GASNod1+/+ mice; however,

infection with H. pylori similarly augmented this effect as 95% of infected INS-

GASNod1-/- mice developed gastric dysplasia by this time-point.

To assess the levels of microbial colonization in each group, we quantified colony-

forming units (CFU) from each infected animal. There were no differences in

colonization between WT and Nod1-/- INS-GAS mice at any time-point (Figure 5A).

Collectively, these data indicate that the increased injury phenotype that develops in

H. pylori-infected Nod1-deficient mice is not an artifact of a single host genetic

background.

Nod1 deficiency increases gastric mucosal cytokine production in H. pylori-infected

INS-GAS mice

We next sought to elucidate the mechanisms through which loss of Nod1 resulted in

increased inflammation and pre-malignant lesions among H. pylori-infected INS-

GAS mice by quantifying Nod1-mediated immune effectors within uninfected or

infected gastric tissue. Similar to our results in C57BL/6 mice (Figure 2A, 2B, 2C,

Supplemental Tables 2-4), infection with H. pylori induced an increase in the levels

of chemokines, as well as Th1, Th2, and Th17 cytokines in both wild-type and Nod1

deficient mice when compared to uninfected controls and these changes were

augmented within the context of Nod1 deficiency (Figure 6A, 6B, 6C, 6D,

Supplemental Tables 10-13). For ease of presentation, we have included cytokines

and chemokines in Figure 6 that 1) were statistically different in terms of levels of

expression between Nod1+/+ and Nod1-/- mice and 2) could be classified as either

chemokines or Th1, Th2, or Th17 cytokines. The complete sets of data are

contained in Supplemental Tables 10-13. Because inflammation was more severe in

Nod1 deficient INS-GAS mice at 20 and 40 days post-infection (Figure 5A, 5B), we

focused on selected differences between H. pylori-infected wild-type versus Nod1

deficient mice at these time-points (Figure 6B, 6C). Specifically, at 20 days post-

challenge, levels of IL-12p40 and TNF- were exclusively increased in infected INS-

GASNod1-/- compared to INS-GASWT mice and at 20 and 40 days post-infection, levels

of IL-6 were exclusively increased in the same pattern. There was also a shift

towards more chemokines and cytokines being increased in INS-GASNod1+/+ mice

compared to their Nod1 deficient counterparts as the duration of infection

progressed (Figure 6A, 6B, 6C, 6D). Collectively, these results and results from

C57BL/6 mice demonstrate that loss of Nod1 alters the severity of mucosal damage

as well as the portfolio of chemokine and cytokine expression within H. pylori-

infected gastric mucosa in 2 different murine models of gastric injury. However, the

effects of Nod1 deficiency wane over time, suggesting that Nod1 exerts its effects

predominantly on innate immune responses, which is consistent with our

epithelial:macrophage gastroid co-culture results.

Finally, because H. pylori is a human gastric pathogen, we ascertained the effects of

infection on Nod1-dependent signaling in human gastric epithelial cells. For these

studies, stably transfected human AGS gastric epithelial cells harboring either

NOD1-targeting or control shRNA were used as previously described (22). Inhibition

of NOD1 prior to infection with the H. pylori cag+ strains PMSS1 or 7.13 increased

production of CXCL8 and CXCL2, confirming our data in mouse gastroids that

suppression of Nod1 can augment inflammatory responses to this pathogen (Figure

6E).

Discussion The innate immune system is exquisitely poised to detect and respond to bacteria

residing at mucosal surfaces (38, 39). However, chronic mucosal pathogens have

developed multiple strategies to subvert this facet of the immune response (40). H.

pylori has co-evolved with its cognate human host for over 100,000 years and is

uniquely adapted to survive for decades within the harsh environment of the

stomach (41). This has necessitated the development of mechanisms to induce

inflammation as well as strategies to evade detection and down-regulate the host

immune response.

H. pylori harbors multiple pathogen-associated mucosal patterns (PAMPs) that

interact differently with innate immune effectors than the respective counterparts in

other acute mucosal pathogens. For example, H. pylori FlaA is a non-inflammatory

molecule in terms of its ability to activate TLR5 (42). H. pylori LPS contains an

anergic lipid A core that induces an attenuated TLR4-mediated response (43, 44).

The cag T4SS delivers peptidoglycan into host cells, where it is recognized by

NOD1 (7-11), and although NF-B activation is a prototypical response to NOD1

activation, we and others have shown that pre-activation of NOD1 suppresses

subsequent H. pylori-induced NF-B signaling via activation of a negative feedback

loop, and that deacetylation of peptidoglycan allows H. pylori to evade host

clearance (22, 45-47). More recent work has also indicated that NOD1 may

suppress gastric inflammation in response to H. pylori. Tran et al. used a mouse

model of gastritis to demonstrate that H. pylori promotes the activation of IL-33, a

mediator of Th2 immune responses, via Nod1 signaling (48). Importantly, H. pylori

cag+ strains specifically activated Nod1 in mouse gastric epithelial cells leading to

enhanced levels of IL-33 in gastric mucosa and splenocytes, which was linked with

reduced IFN- responses (48). Our current results are consistent with these data, as

loss of Nod1 in 2 independent models of H. pylori-induced inflammation and injury

augmented damage within the gastric niche. However, there are additional pathways

that can regulate NF-B activation following H. pylori infection which are

independent of NOD1. Gall et al. demonstrated that NF-B activation can be

induced by tumor necrosis factor receptor-associated factor (TRAF)-interacting

protein with forkhead-associated domain (TIFA), and that this occurs independently

from NOD1-mediated NF-B activation (49). Further, NF-B activation following H.

pylori infection occurs in a temporally regulated manner, with TIFA induction

occurring early, which is subsequently followed by NOD1-dependent NF-B

activation (49). Selective activation of these additional pathways under different

experimental conditions may account for differences in our current results when

compared to previous results published by Viala et al. (7). Therefore, future studies

should focus on assessing both NOD1 and TIFA innate immune signaling pathways

in primary organoid systems as well as animal models of infection, utilizing both wild-

type H. pylori cag+ strains and mutant strains with defective cag secretion systems,

to precisely elucidate the combinatorial contributions of these constituents to H.

pylori pathogenesis.

In this study, Nod1-/- genotypes did not alter the microbial phenotype of H. pylori

output derivatives in terms of T4SS function. However, Nod1 deficient mice

developed more severe inflammation compared to wild-type mice when colonized

with H. pylori strains harboring a functional cag T4SS. Using a multiplex cytokine

array to examine distinct host immune effectors that may contribute to this increase

in inflammation, we observed that, as expected, H. pylori infection broadly increased

levels of Th1, Th2, and Th17 cytokines, and these changes were augmented in the

presence of Nod1 deficiency. However, differences stratified on the basis of Nod1

genotype waned over time. To dissect this further, we utilized an innovative

reductionist system in which the effects of H. pylori on WT or Nod1-/- epithelial cells

alone or epithelial cells co-cultured with macrophages of varying Nod1 genotype

could be ascertained. We determined that loss of Nod1 in both epithelial cells and

macrophages augmented the production of pro-inflammatory cytokine production in

response to H. pylori infection, further supporting the premise that NOD1 primarily

alters innate immune responses to this pathogen. To more fully define the respective

contributions of each of these cellular constituents, however, would require

eliminating the macrophage and the gastric epithelial cell component of Nod1-

dependent signaling, both individually and in combination. This could be done by

crossing Nod1-floxed mice and Foxa3-Cre mice (which directs expression of Cre

recombinase to gastric epithelium) (50) in conjunction with selective depletion of

macrophages using agents such as clodronate liposomes and our current results

have provided an important framework for these future studies.

In addition to differences in cytokine levels between Nod1+/+ and Nod1-/- mice, we

also found differences in the levels of certain cytokines when we compared C57BL/6

to INS-GAS FVB/N mice. These results likely reflect differences in the genetic

backgrounds of the mice under study as well as the presence of hypergastrinemia,

which is inherent to INS-GAS mice bearing the human gastrin transgene (51).

Gastrin exerts growth factor-like effects on gastric epithelial cells, which may induce

the production of certain chemokines and cytokines (51). Further, INS-GAS mice are

on a FVB/N genetic background which has previously been shown to augment the

risk for carcinogenesis when compared to C57BL/6 mice. Specifically, this may be

due to a polymorphism within the Ptch1 gene, which encodes an inhibitory receptor

for ligands of the Hedgehog gene family (52). Within the context of our results, it was

notable that, among others, expression levels of IL-9 were down-regulated in INS-

GAS Nod1-/- mice when compared to C57BL/6 Nod1-/- mice. IL-9 is a cytokine

produced by Th9 cells, a subpopulation of CD4+ T cells, and several studies have

demonstrated that IL-9 suppresses tumor growth (53, 54). Thus, decreased levels of

IL-9 in H. pylori-infected INS-GAS Nod1-/- mice may contribute to the enhanced

carcinogenic phenotype seen in these mice following infection with this pathogen.

Alterations in the composition of functional macrophage phenotypes within a specific

inflamed niche can significantly alter the risk for carcinogenesis (55). M1 (classically

activated) macrophages clear pathogens via intracellular microbicidal activity and by

secreting inflammatory mediators that promote a Th1-type response, while

simultaneously dampening Th2-type responses (55). M2 (alternatively activated)

macrophages promote wound healing by secreting components of the extracellular

matrix and anti-inflammatory effectors that promote Th2-directed responses while

dampening Th1 responses (5). Mregs (regulatory macrophages) are also anti-

inflammatory, but fail to deposit extracellular matrix (55). Our results utilizing

epithelial gastroid:macrophage co-cultures with or without H. pylori revealed that

NOD1 likely plays a role in macrophage polarization as Nod1 WT macrophages co-

cultured with epithelial cells developed a profound M1 phenotype following exposure

to H. pylori compared to a mixed M1/M2 phenotype exhibited by infected Nod1-/-

macrophages. Such a hybrid phenotype may render Nod1 deficient macrophages

ineffective in dampening respective Th1- or Th2-directed responses, thereby leading

to a more severe pattern of global inflammation; our current results provide an

important framework for defining such mechanisms in future work. Thus, the

capacity of NOD1 to regulate macrophage phenotypes may also contribute to the

ability of H. pylori to evade host immune clearance.

In conclusion, this study demonstrates that loss of NOD1 augments inflammatory

responses to H. pylori within the context of gastric carcinogenesis. NOD1 may exert

its restrictive role by altering macrophage polarization, thereby leading to immune

evasion and microbial persistence. These studies lay the foundation for further

exploration into the role of NOD1-H. pylori interactions in human hosts and suggest

that manipulation of NOD1 may represent a novel strategy to prevent or treat

pathologic outcomes induced by H. pylori infection.

Acknowledgments

Financial Support: NIH R01-DK58587, R01-CA77955, P01-CA116087, P30- DK058404, P01-CA028842

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Figure Legends

Figure 1. Nod1 suppresses the early inflammatory response to H. pylori but

does not alter microbial virulence phenotypes

A. Gastric mucosal inflammatory scores and colonization density for C57BL/6 WT

(white bars) and Nod1-/- (Gray bars) male (M) and female (F) mice infected with or

without H. pylori strain PMSS1 for 2 days (WT uninfected [n=6, 3M/3F] and infected

[n=9, 5M/4F]; Nod1-/- uninfected [n=9, 5M/4F] and infected [n=9, 5M/4F]), 20 days

(WT uninfected [n=10, 5M/5F] and infected [n=11, 5M/6F]; Nod1-/- uninfected [n=8,

7M/1F] and infected [n=9, 4M/5F]), and 90 days (WT uninfected [n=7, 4M/3F] and

infected [n=10, 5M/5F]; Nod1-/- uninfected [n=10, 5M/5F] and infected [n=11, 5M/6F]).

Bars represent mean inflammation scores (left axis) or CFU/g tissue (right axis) ±

SEM. *: p≤0.05. B. Representative histologic images from uninfected or H. pylori-

infected WT and Nod1-/- mice. C. Western blot analysis for CagA (phosphorylated: P-

CagA; total: T-CagA) in AGS cells co-cultured with output H. pylori strains isolated

from C57BL/6 WT and Nod1-/- mice infected for 20 and 90 days. Bars graphs

represent densitometric index of P-CagA over T-CagA, which reflects the intensity of

CagA translocation. An H. pylori PMSS1 isogenic pgdA- mutant strain (22) was

included as a bacterial control to ensure that altering H. pylori peptidoglycan did not

reduce CagA translocation.

Figure 2. Loss of Nod1 alters cytokine production in gastric mucosa of

C57BL/6 mice following H. pylori infection

Expression of chemokines and cytokines in gastric mucosa of C57BL/6 WT and

Nod1-/- mice infected with H. pylori strain PMSS1 for 2 (A), 20 (B), and 90 days (C).

Bars represent mean±SEM. Light shaded bars: upregulated in Nod1-/- mice; dark

shaded bars: upregulated in WT mice. *: p≤0.05; **: p≤0.01; ***: p≤0.001.

Figure 3. H. pylori differentially alters cytokine production and MAP kinase

activation in gastroids in a Nod1 dependent manner

RT2 profiler PCR array analysis for NF-B gene targets in primary gastric epithelial

cell organoids generated from C57BL/6 WT and Nod1-/- uninfected mice; control

gastroids were uninfected ex vivo (A) or co-cultured with H. pylori strain PMSS1 for

6 (B) or 24 (C) hours at MOI of 30. Black circles represent genes up-regulated and

gray circles represent genes down-regulated in gastroids from Nod1-/- mice

compared with WT mice. Solid line represents the linear regression of the WT

samples, and dotted lines represent 95% confidence intervals. D. Protein levels of

KC (Cxcl1) in supernatants of primary gastric epithelial cell organoids from WT or

Nod1-/- mice co-cultured with H. pylori PMSS1 for 24 hours at MOI of 30. Bars

represent mean±SEM. **: p≤0.01; and ***: p≤0.001. E. Immunoflourescence for

phospho-c-Jun nuclear translocation in primary gastric cell monolayers generated

+/+ from C57BL/6 and INS-GAS Nod1 and Nod1-/- uninfected mice, that were then

infected with or without H. pylori strains PMSS1 or 7.13 (MOI 30) for 1 hour. Green:

phospho-c-Jun; blue: DAPI; and red: phalloidin; 60X magnification. F. Quantification

of nuclear c-Jun is shown adjacent to representative images. *: p<0.05; **: p≤0.01;

***: p≤0.001; ****: p<0.0001.

Figure 4. Nod1 regulates cytokine production in multiple innate immune cells

following H. pylori infection

Cytokine and chemokine expression as determined by multiplex bead array from

bone marrow (BM)-derived macrophages harvested from C57BL/6 WT or Nod1-/-

mice placed in the lower chamber of a transwell system in co-culture with primary

gastric epithelial cell (GEC) monolayers isolated from the same mice placed in the

upper chamber and then infected with H. pylori strain (upper chamber) for 24 hours

at MOI of 30. A. Comparison of H. pylori infected co-cultures of WT BM

macrophages and WT GEC with co-cultures of WT BM macrophages and Nod1-/-

GEC. B. Comparison of H. pylori infected co-cultures of Nod1-/- BM macrophages

and WT GEC with co-cultures of Nod1-/- BM macrophages and Nod1-/- GEC. Dotted

lines represent 95% confidence interval. Black circles represent up-regulated, and

gray circles represent down-regulated chemokines or cytokines. C. Quantification of

macrophage differentiation marker expression by real time RT-PCR using RNA

isolated from bone marrow derived macrophages (bottom chamber) co-cultured in

transwell systems with corresponding gastric epithelial cells (upper chamber),

uninfected or infected with H. pylori (upper chamber) for 24 hours at MOI of 30.

Primer sequences are shown in Supplemental Table 14. Bars represent mean±SEM.

Light bars represent cells derived from C57BL/6 WT mice; dark bars represent cells

derived from C57BL/6 Nod1-/- mice. *: p≤0.05.

Figure 5. Loss of Nod1 accelerates gastric carcinogenesis in mice

A. Gastric mucosal inflammatory scores and colonization density for male INS-GAS

Nod1+/+ mice and INS-GAS Nod1-/- mice infected with or without H. pylori strain

PMSS1 for 2 days (Nod1+/+ uninfected [n=5] and infected [n=5]; Nod1-/- uninfected

[n=5] and infected [n=5]), 20 days (Nod1+/+ uninfected [n=10] and infected [n=10];

Nod1-/- uninfected [n=10] and infected [n=10]), 40 days (Nod1+/+ uninfected [n=5] and

infected [n=6]; Nod1-/- uninfected [n=5] and infected [n=6]), and 90 days (Nod1+/+

uninfected [n=5] and infected [n=8]; Nod1-/- uninfected [n=5] and infected [n=13]).

Bars represent mean inflammation scores (left axis) or CFU/g tissue (right axis) ±

SEM. *: p≤0.05; **: p≤0.01. B. Representative histologic images from uninfected or

H. pylori-infected INS-GAS Nod1+/+ mice and INS-GAS Nod1-/- mice. Higher

magnification insets of dysplastic foci are shown below other histologic images. C.

Frequency of gastric dysplasia in INS-GAS Nod1+/+ and INS-GAS Nod1-/- mice,

uninfected or infected with H. pylori strain PMSS1 for 40 or 90 days. White bars: no

dysplasia; gray bars: dysplasia. ***: p≤0.001; ****: p≤0.0001.

Figure 6. Nod1 deficiency increases gastric mucosal chemokine/cytokine

production in H. pylori-infected INS-GAS mice and human gastric epithelial

cells

Expression of chemokines and cytokines in gastric mucosa of INS-GAS Nod1+/+ and

INS-GAS Nod1-/- mice infected with H. pylori strain PMSS1 for 2 (A), 20 (B), 40 (C),

and 90 days (D). Bars represent mean±SEM. Light shaded bars: up-regulated in

INS-GAS Nod1-/- mice; dark shaded bars: up-regulated in INS-GAS Nod1+/+ mice. *:

p≤0.05; **: p≤0.01. (E) AGS cells stably transfected with either non-targeting shRNA

or shRNA specific for NOD1 were co-cultured with strains 7.13 or PMSS1 at MOI 30

for 6 and 24 hours. Expression levels of CXCL8 (left axis) and CXCL2 (right axis)

were determined by real-time RT-PCR. Primer sequences are shown in

Supplemental Table 14. Data represent mean±SEM from at least 2 experiments.

**:p≤0.01;*:p≤0.05.

Nod1 imprints inflammatory and carcinogenic responses toward the gastric pathogen Helicobacter pylori

Giovanni Suarez, Judith Romero-Gallo, Maria Blanca Piazuelo, et al.

Cancer Res Published OnlineFirst January 29, 2019.

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