Colitis susceptibility in p47 phox−/− mice is mediated by the microbiome
© Falcone et al. 2016
Received: 19 November 2015
Accepted: 24 February 2016
Published: 5 April 2016
Chronic granulomatous disease (CGD) is caused by defects in nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) complex subunits (gp91 phox (a.k.a. Nox2), p47 phox , p67 phox , p22 phox , p40 phox ) leading to reduced phagocyte-derived reactive oxygen species production. Almost half of patients with CGD develop inflammatory bowel disease, and the involvement of the intestinal microbiome in relation to this predisposing immunodeficiency has not been explored.
Although CGD mice do not spontaneously develop colitis, we demonstrate that p47 phox−/− mice have increased susceptibility to dextran sodium sulfate colitis in association with a distinct colonic transcript and microbiome signature. Neither restoring NOX2 reactive oxygen species production nor normalizing the microbiome using cohoused adult p47 phox−/− with B6Tac (wild type) mice reversed this phenotype. However, breeding p47 phox+/− mice and standardizing the microflora between littermate p47 phox−/− and B6Tac mice from birth significantly reduced dextran sodium sulfate colitis susceptibility in p47 phox−/− mice. We found similarly decreased colitis susceptibility in littermate p47 phox−/− and B6Tac mice treated with Citrobacter rodentium.
Our findings suggest that the microbiome signature established at birth may play a bigger role than phagocyte-derived reactive oxygen species in mediating colitis susceptibility in CGD mice. These data further support bacteria-related disease in CGD colitis.
KeywordsChronic granulomatous disease NADPH Reactive oxygen species p47 phox Microbiome Inflammatory bowel disease Colitis Dextran sodium sulfate
Chronic granulomatous disease (CGD) is a genetic immunodeficiency caused by defects in any one of the five subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) complex including gp91 phox (a.k.a Nox2) (CYBB (cytochrome b-245, beta polypeptide)), p47 phox (NCF1 (neutrophil cytosolic factor 1)), p67 phox (NCF2 (neutrophil cytosolic factor 2)), p22 phox (CYBA (cytochrome b-245, alpha polypeptide)), and p40 phox (NCF4 (neutrophil cytosolic factor 4)) . This leads to reduced or absent production of reactive oxygen species (ROS) primarily in phagocytes, which manifests clinically as the early onset of recurrent infections, and marked dysregulation of inflammation [2, 3]. In fact, our recent survey of patients with CGD followed at the National Institutes of Health (NIH) suggests that almost 50 % of these patients suffer from inflammatory bowel disease (IBD) (unpublished data).
It has been established that aberrant interactions between the intestinal microbiota and the immune system may fuel intestinal and systemic inflammation (reviewed in [4, 5]). However, it is unclear whether the absence of phagocyte-derived ROS drives intestinal dysbiosis in either mice or humans and if intestinal dysbiosis is a cause or consequence of CGD gastrointestinal (GI) inflammation. Our objective therefore was to define the contribution of the intestinal microbiota in driving IBD penetrance in the context of CGD as a predisposing monogenetic immunodeficiency.
We induced colitis in p47 phox−/− mice and used bone marrow chimeras, as well as 16S rRNA sequencing in conjunction with microbiome standardization techniques, to examine the contribution of phagocyte-derived ROS in driving intestinal dysbiosis and to determine the role of the microbiome in modulating colitis susceptibility in CGD mice. We found that although phagocyte-derived ROS modulates intestinal microbiomic and transcriptomic signatures, the intestinal microbiota established at birth has a greater impact on colitis susceptibility in p47 phox−/− mice then the absence or presence of phagocyte-derived ROS.
p47phox−/− mice do not spontaneously develop colitis and their neutrophils do not produce ROS
Untreated p47 phox−/− mice were monitored daily for 16 months. There were no mortalities, and histological examination of colons showed no evidence of subclinical gastrointestinal disease. p47 phox−/− colons were found to be similar to those of 8-week-old B6Tac wild-type (WT) mice (Additional file 1: Figure S1). We confirmed that neutrophils isolated from p47 phox−/− mice do not produce ROS by performing dihydrorhodamine (DHR) oxidation assays before and after stimulation with phorbol 12-myristate 13-acetate (PMA) (Additional file 2: Figure S2).
p47phox−/− mice have increased susceptibility to DSS colitis
p47phox−/− mice have a distinct colonic transcript profile that is not associated with a pattern of leukocyte infiltration during DSS colitis
Increased DSS colitis susceptibility in p47phox−/− mice is not reversed by restoration of NOX2-mediated ROS production
p47phox−/− mice have a distinct intestinal microbiome signature before and after DSS colitis
p47phox−/− mice cohoused with B6Tac mice maintain increased susceptibility to DSS colitis
Homogenizing the intestinal microbiome at birth decreases DSS and Citrobacter rodentium colitis susceptibility in p47phox−/− mice
Deficiencies in any of the five subunits of the NOX2 complex cause CGD, and almost half of all CGD patients will develop IBD (unpublished data). Genome-wide association studies have associated the NOX2 subunit genes with IBD and very early-onset IBD [7, 8], suggesting that an understanding of the mechanisms underlying CGD colitis may have broader applications than to this rare immunodeficiency alone. We examined intestinal inflammation in p47 phox−/− mice using DSS colitis. Surprisingly, restoring phagocyte ROS production did not reduce DSS colitis susceptibility in p47 phox−/− mice. 16S rRNA fecal sequencing confirmed that homozygously bred p47 phox−/− and B6Tac mice have distinct microbiome signatures, which can be normalized by cohousing or heterozygous breeding. Cohousing and littermate control experiments uncovered a previously unrecognized contribution of the intestinal microbiome to DSS and C. rodentium colitis susceptibility, which is critically established before weaning.
Unlike humans with CGD, p47 phox−/− mice do not spontaneously develop colitis. We induced colitis using DSS, which denudes the colonic epithelium allowing for bacterial translocation and engaging the innate immune system . We found that the absence of p47 phox in this setting was not only associated with increased colitis severity but also mortality. Likely causes of death include a combination of dehydration, malnutrition, GI blood loss, bacteremia, and cytokine elevation. Our observed increase in colitis susceptibility in p47 phox−/− mice is supported by a previous study, which demonstrated increased weight loss, colitis severity, and leukocyte infiltration, as well as localized and systemic cytokine production in p47 phox−/− mice .
The absence of p47 phox in the mouse colon was associated with a specific gene expression signature before and after the induction of DSS colitis. The gene expression signature post-DSS discriminated p47 phox−/− from WT mice. It also highlighted the potential roles of chemokines, inflammatory cytokines, and candidate pathways such as PPARγ and IL1α/IL1β. We were limited by the use of a custom-designed gene probe panel, which introduced gene selection bias into the analysis. Since our objective was to detect discriminative signatures, gene expression in whole colons was examined, instead of in specific cell types.
Previous cohousing studies have demonstrated that the intestinal microbiome plays an important role in the development and transfer of DSS colitis susceptibility [11, 12]. It is therefore imperative that the contribution of the intestinal microbiota be teased out from the host genetic predisposition to DSS-induced inflammation. Unsurprisingly, p47 phox−/− mice had a distinct microbiome signature at baseline, characterized by more abundant Akkermansia muciniphila. This gram-negative intestinal mucolytic  is reduced in humans with IBD [14, 15]. Moreover, feeding DSS-induced mice A. muciniphila-derived extracellular vesicles reduces IBD severity . After DSS colitis induction, A. muciniphila was no longer more abundant in p47 phox−/− mice. Of note, segmented filamentous bacteria, which in mice are correlated with increased secretion of pro-inflammatory IL-17 and decreased regulatory T cells , were not differentially abundant in any of the experimental groups studied in each experimental setting. Overall, DSS induction led to somewhat less microbial community structure dissimilarity between mouse genotypes, as well as less bacterial diversity. Cohousing p47 phox−/− mice with B6Tac mice, and thus exposing p47 phox−/− mice to WT fecal microbiomes, did not protect p47 phox−/− mice from developing severe colitis. However, susceptibility to both DSS and C. rodentium colitis was significantly reduced when examining heterozygously bred p47 phox−/− mice and their littermate controls. As expected, 16S rRNA sequencing confirmed that cohousing and heterozygous breeding were effective in normalizing the microbiota between p47 phox−/− and B6Tac mice. Moreover, in both settings, the strains of translocating bacteria became similar between experimental groups. Nevertheless, it should be noted that 16S rRNA sequencing analysis might not capture discrete differences between bacterial strains that do not vary in abundance between compared experimental groups.
In contrast, restoration of hematopoietic p47 phox using bone marrow chimeras did not decrease susceptibility to DSS colitis in p47 phox−/− mice, nor did transplanting p47 phox -deficient cells into WT mice increase their susceptibility to DSS colitis. A potential limitation of these bone marrow chimeras is the radiosensitivity of the components in the organ of interest. We obtained greater than 94 % donor chimerism for hematopoietic cells in the lamina propria. Nevertheless, the colon harbors many stromal cells with immunological functions, as well as stem cells, which are not derived from the hematopoietic compartment . Therefore, future studies will need to examine selective depletion of p47 phox in specific colonic cell populations. Moreover, although humans with CGD may have improved colitis following hematopoietic stem cell transplantation , there may be differences in either the intestinal microbiota or p47 phox -NOX complex interactions in non-hematopoietic colonic cells, which may explain the species-specific differences in colitis remission post-bone marrow transplantation.
p47 phox -deficient mice have increased DSS-induced intestinal inflammation in association with a unique gene expression signature, which is profoundly affected by the intestinal microbiomic signature established at birth. The CGD genotype shapes the intestinal microbiota, which in turn drives IBD penetrance in this immunodeficiency. Future clinical studies should include characterizing the fecal microbiome in CGD patients at birth in order to elucidate potential candidates for bacteriotherapy.
p47 phox -deficient mice, B6.129S2-Ncf1 tm1shl N14 (p47 phox−/− ), generated as previously described , were backcrossed onto the C57BL/6NTac (Taconic Farms) background for 14 generations and inbred in our facility. C57BL/6 NTac (B6Tac) and CD45+ congenic B6.SJL (B6Tac-CD45.1+) mice were purchased from Taconic Farms and inbred in our facility for ≥2 generations. For littermate experiments, p47 phox−/− mice were bred with B6Tac mice to generate heterozygous (p47 phox+/− ) mice, which were bred together to generate littermate p47 phox−/− and WT mice. Of note, DSS colitis susceptibility in p47 phox+/− mice is comparable to that observed in WT mice (data not shown). For cohousing experiments, 4-week-old p47 phox−/− female weanlings were placed in fresh cages with female age-matched B6Tac mice (1:1 ratio; two mice per cage) for 5 weeks prior to DSS colitis induction while mice remained cohoused. Mice were gender and age (8–11 weeks) matched for each experiment. All mice were housed in aseptic, specific pathogen-free conditions in the same room of the same animal facility at the National Institutes of Allergy and Infectious Diseases (NIAID) (Bethesda, MD), and the NIAID Animal Care and Use Committee approved all experiments. Unless otherwise specified, 3–10 mice were used per experimental group, and all experiments were performed at least twice.
Mice were administered filter-sterilized 3.5 % (w/v) DSS (m.w. 36,000–50,000; MP Biomedicals)-supplemented drinking water ad libitum for 7 days, followed by 1 day of regular autoclaved water. Mice were monitored daily for weight, disease activity, and survival. The DAI was scored as previously described . Briefly, it consists of the sum of the scores attributed to weight loss (0–4), stool consistency (0, 2, 4), and fecal blood (0, 2, 4) divided by 3. Colons (from anus to ilieo-cecal junction) were harvested at indicated time points for histology and RNA extraction.
C. rodentium infection
C rodentium suspension was prepared by shaking incubation at 37 °C for 4 h in Luria Broth. Bacterial concentration was assessed by absorbance at an optical density of 600 nm and confirmed by plating of serial dilutions. Mice were inoculated by oral gavage with 5 × 109 colony-forming units (CFU) of C. rodentium and monitored for weight and survival. Twelve days post-infection, all mice were euthanized. Sections of spleen and MLNs were analyzed for the presence of viable bacteria. Colons (from anus to ilieo-cecal junction) were harvested on day 12 post-infection for histology. Stool samples were harvested on day 12 and plated on MacConkey agar (Remel) for evaluation of C. rodentium fecal load.
DHR oxidation assay on murine neutrophils
Production of neutrophil-derived ROS was measured using a DHR oxidation assay. Room temperature heparinized blood (300 μL) obtained from B6Tac and p47 phox−/− mice (one male and one female from each strain) was lysed in filtered flow lysis buffer (NH4Cl (0.155 M), KHCO3 (0.01 M), EDTA (0.1 mM) in distilled H2O) for 5 min and resuspended in flow buffer HBSS with 0.1 % BSA and 0.1 M EDTA. Cells were incubated with DHR and catalase for 5 min and stimulated with PMA (Sigma) for 15 min. Cells were analyzed by flow cytometry using a BD Canto II cytometer (BD Biosciences). Data analysis was performed using FlowJo software (Tree Star).
Histology and immunohistochemistry
Formalin-fixed colons were paraffin-embedded, and 5-μm-thick sections were either stained with H&E or processed for IHC. H&E-stained colon sections from DSS-treated mice were scored in a blinded fashion as per a modified version of the method described in . Each section was scored for the following parameters: severity of inflammation (0–3), depth of inflammation/injury (0–3), and crypt damage (0–4). Before adding up the scores for each parameter, each score was multiplied by a factor representing the percentage of tissue involved (X 1 for 0–25 %; X 2 for 26–50 %; X 3 for 51–75 %; X 4 for 76–100 %). Thus, the maximum histological severity score in this model is 40. H&E-stained colon sections from C. rodentium-infected mice were scored in a blinded fashion as per the method described in .
IHC sections were set on poly-l-lysine-coated glass slides, deparaffinized, and rehydrated in graded concentrations of ethanol, followed by heat-induced epitope retrieval. Endogenous peroxidase was blocked using methanol containing 3 % H2O2 for 15–30 min. Slides were blocked with BSA (Sigma) and incubated overnight at 4° with primary antibody to MPO (Abcam), Mac-1 (Novus biological), CD3 (Abcam), or CD138 (BD Pharmingen). Slides were washed with PBS three times and immunolabeled using the ImmPRESS detection system followed by visualization with ImmPACT Dab peroxidase substrate (Vector). Slides were then counterstained with hematoxylin, mounted with Permount (Fisher), and scanned with a ScanScope (Aperio). Quantification of primary antibody staining was performed by counting the number of stained cells per high-power field (hpf) along the thickness of the same distal colon segment for each mouse.
Bacterial translocation and identification
MLNs and spleens were aseptically retrieved from mice at baseline and after DSS or C. rodentium infection. Tissue was homogenized in sterile HBSS, plated onto sheep blood agar plates (Trypticase Soy Agar with 5 % Sheep Blood, Remel), and incubated at 37 °C for 48 h before CFU quantification.
Microbiological identification of isolates was performed using MALDI-TOF MS (Bruker Daltonics) as previously described . For protein extraction, bacterial colonies in sheep blood agar plate were resuspended in 1 ml 70 % ethanol, vortexed for 1 min, and centrifuged at 13,000 rpm for 2 min. The supernatant was removed completely, and the sample was vortexed for 10 s with 50 μl of 70 % formic acid (FA) and 50 μl acetonitrile (ACN). After 2-min centrifugation at 13,000 rpm, 1 μl of supernatant was spotted onto the target plate and overlaid with 2 μl of alpha-cyano-4-hydroxycinnamic acid (α-CHCA). MALDI-TOF MS analysis was performed using a Microflex LT spectrometer (Bruker Daltonics) and the Biotyper version 22.214.171.124. Manufacturer-recommended cutoff scores of ≥2.0 for species-level identification and ≥1.7 for genus-level identification, and >10 % difference of top score from other genera and species were applied.
Distal colons were rinsed in RNAlater (Ambion) and stored at −80 °C in RLT buffer (Qiagen) until RNA isolation. RNA was extracted from homogenized distal colon segments using the RNeasy kit (Qiagen) as per the manufacturer’s protocol. Total RNA (250 ng) was hybridized with reporter and capture probes for a murine custom probe set (Nanostring Technologies) as per manufacturer’s instructions. Samples were prepared on an nCounter Prep station and analyzed on an nCounter Analysis system (Nanostring Technologies). Data were normalized to housekeeping genes and spiked positive controls. Transcript counts less than the mean of the negative control transcripts plus 1 standard deviation (SD) for each sample were considered as background.
Generation of bone marrow chimeras
Femurs and tibias from donor 7- to 8-week-old p47 phox−/− (CD45.2+) and B6Tac congenic SJL (CD45.1+) mice were removed aseptically, and BM was flushed using sterile cold PBS supplemented with 2 mM EDTA. Seven to eight-week-old recipient p47 phox−/− (CD45.2+) and B6Tac-CD45.1+ mice were irradiated with 9 Gy and reconstituted 8 h later with 5 × 106 B6Tac-CD45.1+ cells (B6Tac-CD45.1+ → B6Tac-CD45.1+ and B6Tac-CD45.1+ → p47 phox−/− mice) or p47 phox−/− cells (p47 phox−/− → p47 phox−/− and p47 phox−/− → B6Tac-CD45.1+ mice) by lateral tail-vein injection. Mice were given trimethoprim-sulfamethoxazole-supplemented drinking water for the first 4 weeks of reconstitution before being switched to regular drinking water as previously described . Chimeras were treated with DSS 10 weeks after transplantation.
Prior to DSS administration, reconstitution with congenic BM stem cells to a satisfactory level of chimerism was confirmed by assessing the number of CD45.1+ (B6Tac-CD45.1+) and CD45.2+ (p47 phox−/− ) leukocytes in the blood and colon using flow cytometry. Colon cell suspensions were generated as described . Cells from blood and colon suspensions were stained with the Live/Dead Fixable Blue staining kit (Invitrogen), followed by Fc blockade with anti-CD16/32 (2.4G2) (eBioscience), followed by staining with antibodies to CD3 (clone 17A2), CD19 (clone 1D3), CD45 (clone 30-F11), CD45.1 (clone A20), CD45.2 (clone 104), MHCII, F4/80 (clone BM8), CD11b (clone M1/70), Ly6C (clone AL-21), Ly6G (clone 1A8) (all antibodies from eBioscience except Ly6C and Ly6G are from BD biosciences). Samples were analyzed on a FACS Fortessa cytometer (BD Biosciences), and data analysis was performed with FlowJo software (Tree Star). In both the blood and the colon, the proportion of donor-derived monocytes, neutrophils, and B cells exceeded 95 %, whereas that of T cells exceeded 80 %.
Plasma cytokine measurement
Cytokines were measured in mouse plasma using the Bio-Plex® Multiplex Immunoassay based on the Luminex xMAP technology, and data were analyzed using the Bio-Plex® Manager software (Bio-Rad).
Fecal microbiome analysis
DNA was extracted from sterilely excised stool pellets using PowerSoil® DNA Isolation Kit (MO BIO Laboratories) as per manufacturer’s instructions. The V1-3 region of the 16S rRNA gene was amplified and sequenced using the 454 GS FLX pyrosequencing platform (Roche). The 16S rRNA gene sequences were processed using the mothur software  to retain high-quality reads prior to analysis. Preprocessing steps included denoising, trimming, alignment to SILVA 16S rRNA sequence database, pre-clustering, and chimera removal. Sequences were classified using a naive Bayesian classifier trained against a 16S rRNA gene training set provided by the Greengenes Database (May 2013 release). Sequences were then clustered into OTUs using a 3 % distance cutoff with the average neighbor-clustering algorithm. The phyloseq R package  was used to plot diversity and richness (Shannon diversity and inverse Simpson calculators, respectively) within samples. To estimate dissimilarity in community structure between pairs of samples, we used the mothur implementation of θ YC calculator, which uses the OTU frequency data and subsequently ran parsimony tests (P test) to determine significance of differences. UniFrac metrics were calculated using neighbor-joining phylogenetic trees generated using the non-heuristic neighbor-joining algorithm implemented in the software program Clearcut  and used as input for the PCoA plots. LEfSe was used to identify features, in this case OTUs, that were statistically different among experimental groups. For each pair of sample groups compared, the non-parametric factorial Kruskal-Wallis sum-rank test detected OTUs with significant differential abundance for each class. A significance alpha of 0.05 and an LDA effect size threshold of 2 were used for all biomarkers reported. A p < 0.05 was considered significant. The biomarkers were sorted by LDA score, and those with values in the top quartile were selected to be included in the heat maps. The values used in the heat maps are the relative abundance of the OTUs identified as biomarkers . All graphs were generated using R software (version 3.0.2).
The Mann-Whitney U test or, where applicable, a log-rank test (Mantel-Cox) was used to compare values between groups and time points using Prism 6 software (GraphPad Software). A p < 0.05 was considered significant. Unless otherwise indicated, numerical values represent medians, and error bars represent inter-quartile range. For gene expression analyses, the differences between experimental groups were assessed by two-tailed Student’s t tests with Welch approximation using MultiExperiment Viewer (MeV) software.
All experiments were conducted in accordance with guidelines set forth by the Guide for the Care and Use of Laboratory Animals under a protocol approved by the Animal Care and Use Committee of the NIAID in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited animal facility.
Availability of supporting data
The raw sequence files supporting the results of this manuscript are available in the NCBI Sequence Read Archive (SRA) under accession number SRP067040.
- Ccl3 :
chemokine (C-C motif) ligand 3
chronic granulomatous disease
- Cxcl2 :
chemokine (C-X-C motif) ligand 2.
dextran sulfate sodium
- G-csf :
granulocyte-colony stimulating factor
hematoxylin and eosin
inflammatory bowel disease
linear discriminant analysis effect size
- MALDI-TOF MS:
matrix-assisted laser desorption ionization-time of flight mass spectrometry
mesenteric lymph node
nicotinamide adenine dinucleotide phosphate
National Institutes of Allergy and Infectious Diseases
NADPH oxidase 2
operational taxonomic unit
- P test:
principal component analysis
principal coordinate analysis
phorbol 12-myristate 13-acetate
reactive oxygen species
We would like to acknowledge the assistance of the members of the 14B South Animal Facility at the NIH, in particular Danielle N. Mitchell and Vaneesha Bradford. We would like to thank Dr. Frida Stock (CC, NIH) for her assistance with performing the MALDI-TOF MS experiments and Ashley McMichael for her assistance with the C. rodentium experiments. We would also like to acknowledge Dr. Thomas Leto (NIAID, NIH), Dr. Yasmine Belkaid (NIAID, NIH), Dr. Brian Kelsall (NIAID, NIH), Dr. Brian Janelsins (NIAID, NIH), Dr. Prabha Chandrasekaran (University of Maryland), and Tu-Ahn Pham and Dr. Trevor Lawley (Wellcome Trust Sanger Institute, Cambridge, UK) for their scientific guidance.
This project was supported by the Division of Intramural Research, the NIAID, NIH, and in part with federal funds from the National Cancer Institute, NIH, under Contract No. HHSN261200800001E.
The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
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