T cell-intrinsic role for Nod2 in protection against Th17-mediated uveitis

Mutations in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) cause Blau syndrome, an inflammatory disorder characterized by uveitis. The antimicrobial functions of Nod2 are well-established, yet the cellular mechanisms by which dysregulated Nod2 causes uveitis remain unknown. Here, we report a non-conventional, T cell-intrinsic function for Nod2 in suppression of Th17 immunity and experimental uveitis. Reconstitution of lymphopenic hosts with Nod2−/− CD4+ T cells or retina-specific autoreactive CD4+ T cells lacking Nod2 reveals a T cell-autonomous, Rip2-independent mechanism for Nod2 in uveitis. In naive animals, Nod2 operates downstream of TCR ligation to suppress activation of memory CD4+ T cells that associate with an autoreactive-like profile involving IL-17 and Ccr7. Interestingly, CD4+ T cells from two Blau syndrome patients show elevated IL-17 and increased CCR7. Our data define Nod2 as a T cell-intrinsic rheostat of Th17 immunity, and open new avenues for T cell-based therapies for Nod2-associated disorders such as Blau syndrome.


Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research Laboratory animals A power analysis determined that for in vivo studies, a group size of n=6 gives us statistical power to detect a mean different of 1.33 times the standard deviation, especially considering the variation that is inherently involved in the experimental mouse model of uveitis. Due to interanimal variability in certain experiments and/or modest treatment effects, then we combined data from 2 or more independently performed studies in order to have a great statistical power.
Data was rarely excluded. In some odd cases, an individual data point was excluded based on the statistical definition of it being an outlier (which was defined as 2-times the standard deviation above or below the mean). There were also situations where a data point was excluded as the animal became sick and was humanely euthanized and removed from the study.
All experiments were replicated independently on a separate group of mice (the number of replicated experiments are defined within the figure legends).
Mice were randomly assigned to treatment groups based on their genotype.
Uveitis (both clinically and histologically) was evaluated in a masked fashion as indicated in the Methods section. Since all mice were assigned a code at the time of immunization for the purpose of repeated clinical evaluation, the collection of samples for in vitro experimentation were also often carried out in a masked fashion.
Antibodies used for flow cytometry were validated via relevant citations and inclusion of positive and negative controls. In all cases validation statements are also available on the manufacture's website. Other than flow cytometry, antibodies were not used in any other experiments to validate.
Mice of the following strains were used in this study: C57Bl/6J and B10.RIII background strains as well as genetically modified mice that lack the indicated genes and/or contain TCR-Transgene. All mice were bred individually in specific pathogen-free (SPF) conditions at the VA Portland Health Care System, where they were maintained at 21C on 12 h light-dark cycle (6am to 6pm) and given free access to food and water. Since we do not observe a sex-bias for males vs. females in the Nod2-associated phenotype both genders were used experimentally (in a matched manner) between the ages of 6-10 wks.
The two related patients with Blau syndrome (confirmed heterozygous CARD15c.1147G->A mutation, leading to pGlu383Lys mutation) included a mother (Blau-2, 48-year-old female) and son (Blau-1, 17-year-old male), who had onset of disease at ages 15 and 9, respectively. Blau-1 had been previously treated with methotrexate and was on adalimumab to control dermatitis and arthritis at the time of blood draw. Blau-2 had been on adalimumab and infliximab in the past and was on abatacept, methotrexate, prednisone (5-15 mg daily), and alendronate to control arthritis, uveitis, and nodular skin lesions with granulomatous inflammation (confirmed by biopsy) at the time of blood draw.
There was no self-selection bias. The two patients with Blau syndrome were selected based on their previously confirmed mutation in NOD2 (heterozygous CARD15c.1147G->A mutation, leading to pGlu383Lys mutation) and their symptoms--both of which defined them as having Blau syndrome. Patients were treated at University of Minnesota were they were enrolled in this research study with their treating physicians Drs. Vehe and Binstadt. Conceivably, given the stated criteria for participation including confirmed Blau Syndrome and that they were patients of Dr. Vehe or Binstadt this presents a potential selection bias. This selection bias could impact the results if the two donors do not reflect the general Blau community. The healthy controls were gender and age-matched accordingly.
Studies were carried out under protocols approved by the Institutional Review Boards of the University of Minnesota and Oregon Health & Science University.
For mouse cells: Eyes were enucleated and the intraocular lens removed. Pineal glands were removed from the skull cap. Single-cell suspensions were prepared from eyes or pineal glands by collagenase-digestion (1 mg/ml Clostridium histolyticum Collagenase D, Roche) for 40 min at 37C and sequential filtration through a 70-and 40-µm strainer as described60. Single cell suspensions from spleens were prepared from immunized mice, and erythrocytes were lysed using red blood cell lysis buffer (Sigma) For human samples: Human CD4+ T cell stimulations were carried out on viable frozen PBMCs from Blau syndrome patients vs. healthy, gender-matched controls. Peripheral blood was collected into BD Vacutainer ® CPT Mononuclear Cell Preparation tubes (Sodium Citrate) and PBMCs were isolated per manufacturer's instructions (BD Bioscience) within 24 h of collection. Cells were subsequently processes for flow cytometry.
Cells were analyzed on an LSRFortessa (BD Biosciences).
FlowJo (Becton, Dickinson, & Company, Franklin Lakes, NJ, USA) was used for analysis, allowing compensations for spectral overlaps within each sample.
Cells were stained with live/dead AQUA (ThermoFischer). Dead cells were excluded based on viability dye staining so that only live cell events were collected.
Following fixation (4% paraformaldehyde), cells were analyzed on an LSRFortessa (BD Biosciences). Dead cells were excluded based on viability dye staining so that only live cell events were collected. FlowJo 10.0 (Becton, Dickinson, & Company, Franklin Lakes, NJ, USA) was used for analysis, allowing compensations for spectral overlaps within each sample. Gating used defined criteria based on control samples stained with corresponding isotype control antibodies (IC Ab) and equal number of total live events were collected. Live cells were gated from forward and side scatter plots in identical fashion for each group, after which frequencies and numbers of leukocyte subpopulations were determined from CD45+ gated cells.