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Originally published online as doi:10.1189/jlb.0108015 on May 21, 2008

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(Journal of Leukocyte Biology. 2008;84:529-536.)
© 2008 by Society for Leukocyte Biology

Activation of NOD2 in vivo induces IL-1β production in the eye via caspase-1 but results in ocular inflammation independently of IL-1 signaling

H. L. Rosenzweig*,1, T. M. Martin*,{dagger}, S. R. Planck*,{ddagger},§, K. Galster*, M. M. Jann, M. P. Davey{dagger},{ddagger},¶, K. Kobayashi||, R. A. Flavell** and J. T. Rosenbaum*,{ddagger},§

* Departments of Ophthalmology,
{dagger} Molecular Microbiology and Immunology,
{ddagger} Medicine, and
§ Cell and Developmental Biology, Oregon Health and Science University, Portland, Oregon, USA;
Veterans Affairs Medical Center, Portland, Oregon, USA;
|| Department of Cancer Immunology and AIDS, Harvard Medical School, Boston, Massachusetts, USA; and
** Department of Immunology, Yale School of Medicine, New Haven, Connecticut, USA

1 Correspondence: Oregon Health & Science University, 3181 S.W. Sam Jackson Park Rd., Mail Stop: L467 IM, Portland, OR 97239, USA. E-mail: rosenzwh{at}ohsu.edu


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ABSTRACT
 
Nucleotide-binding and oligomerization domain 2 (NOD2) belongs to the emerging Nod-like receptor (NLR) family considered important in innate immunity. Mutations in NOD2 cause Blau syndrome, an inherited inflammation of eye, joints, and skin. Mutations in a homologous region of another NLR member, NALP3, cause autoinflammation, wherein IL-1β plays a critical role. Here, we tested the hypothesis that IL-1β is a downstream mediator of NOD2-dependent ocular inflammation. We used a mouse model of NOD2-dependent ocular inflammation induced by muramyl dipeptide (MDP), the minimal bacterial motif sensed by NOD2. We report that MDP-induced ocular inflammation generates IL-1β and IL-18 within the eye in a NOD2- and caspase-1-dependent manner. Surprisingly, two critical measures of ocular inflammation, leukocyte rolling and leukocyte intravascular adherence, appear to be completely independent of IL-1 signaling effects, as caspase-1 and IL-1R1-deficient mice still developed ocular inflammation in response to MDP. In contrast to the eye, a diminished neutrophil response was observed in an in vivo model of MDP-induced peritonitis in caspase-1-deficient mice, suggesting that IL-1β is not essential in NOD2-dependent ocular inflammation, but it is involved, in part, in systemic inflammation triggered by NOD2 activation. This disparity may be influenced by IL-1R antagonist (IL-1Ra), as we observed differential IL-1Ra levels in the eye versus plasma at baseline levels and in response to MDP treatment. This report reveals a new in vivo function of NOD2 within the eye yet importantly, distinguishes NOD2-dependent from NALP3-dependent inflammation, as ocular inflammation in mice occurred independently of IL-1β.

Key Words: transgenic/knockout mice • cytokines


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INTRODUCTION
 
Elucidation of the pathogenesis of NALP3-dependent autoinflammatory diseases has led to remarkably successful therapy for these syndromes in addition to new perspectives about the molecular processes underlying these illnesses as well as gout [1 2 3 ]. Recent data have unveiled the role of NALP3 (also referred to as cryopyrin) as a crucial component of the cytosolic caspase-activating platform, called an inflammasome. These observations have shed light onto the mechanisms of IL-1β processing and release. IL-1 family members, such as IL-1β and IL-18, play important roles in host defense, immune regulation, and inflammation. The importance of the inflammasome in their regulation is derived from studying the biological effects of mutations in the gene encoding NALP3 that causes a triad of syndromes: Muckle-Wells syndrome, neonatal onset multisystem inflammatory disease, and familial cold urticaria [4 5 6 ]. These autoinflammatory syndromes, collectively called cryopyrin-associated periodic syndromes (CAPS), are characterized by periodic fevers and multi-organ inflammation as a result of excessive IL-1β production [7 ]. Each of these syndromes responds extremely well to the IL-1R antagonist (IL-1Ra), anakinra [8 ]. More recently, insights from the NALP3 inflammasome prompted preliminary studies that have demonstrated the effectiveness of the IL-1Ra anakinra in the treatment of gout [9 ].

NALP3 is a crucial component of the inflammasome in the activation of caspase-1 by way of its interactions with apoptosis-associated speck-like protein [3 , 10 ]. Caspase-1 in turn mediates the proteolytic cleavage of the pro-forms of IL-1β and IL-18 to their secreted, active forms. The essential role for NALP3 in inflammasome activation and IL-1β production in response to infectious agents has been demonstrated in NALP3-defienct mice [1 , 3 , 11 ]. In the case of gouty crystals, the NALP3-casapse-1 inflammasome is necessary for IL-1β and IL-18 production. Importantly, deficiency in IL-1R1 prevented neutrophil accumulation in this mouse model of gouty, crystal-induced peritonitis [11 ].

As part of a subfamily of the larger nucleotide-binding and oligomerization domain (Nod)-like receptor (NLR) family, NALP3 shares structural homology with other family members with respect to its C-terminal leucine-rich repeat (LRR) domain and a centrally located NOD [12 ]. In addition to NALP3, other members of the NOD family have been implicated in regulation of the inflammasome [7 , 10 , 13 , 14 ], supporting a role for the NLR family in shaping innate inflammatory responses. In addition, several members of the NLR family, including NOD2, can cause inheritable, autoinflammatory syndromes. In the case of NOD2, point mutations result in an inherited inflammatory disease called Blau syndrome [15 , 16 ], which is characterized by multi-organ, granulomatous inflammation predominantly within the eye, joints, and skin [17 , 18 ]. Polymorphisms in the LRR domain of NOD2 have also been associated with increased risk of developing Crohn’s disease, a granulomatous bowel inflammation [19 ], which can also be associated with intraocular inflammation. Notably, R260W, the most common pathogenic mutation in NALP3, directly corresponds to mutations R334W and R334Q within the NOD domain of NOD2 that cause Blau syndrome [20 ]. Thus, by analogy, it has been proposed that NOD2 may function like NALP3 to activate the inflammasome and that like the NALP3-autoinflammatory syndromes, caspase-1 and IL-1β might also play central roles in Blau syndrome [12 , 21 ].

Although the precise mutations of NOD2 responsible for Blau syndrome have been identified, the mechanisms by which those mutations induce inflammation are largely unknown. Accordingly, we have sought to investigate the function of NOD2 within the eye and how it might relate to the initiation of uveitis or ocular inflammation, which is a predominate characteristic of Blau syndrome. We have previously characterized a mouse model of NOD2-dependent inflammation in the eye that is induced by local injection of muramyl dipeptide (MDP) [22 ], a component of bacterial peptidoglycan and the minimal motif responsible for NOD2 activation [23 ]. Local MDP treatment results in an increased intravascular cellular response within the iris and a leukocyte infiltration within the aqueous, which mainly involves neutrophils. Here, we tested the hypotheses that activation of NOD2 results in IL-1β production via a caspase-1-dependent mechanism and that IL-1β and caspase-1 contribute to MDP-induced ocular inflammation. We report that MDP generates IL-1β within the eye in a NOD2- and caspase-1-dependent manner. Surprisingly, two critical measures of MDP-induced ocular inflammation, leukocyte rolling and leukocyte intravascular adherence, appear to be completely independent of IL-1 signaling effects or caspase-1. In contrast to the eye, a diminished neutrophil response was observed in an in vivo model of MDP-induced peritonitis in caspase-1-deficient mice. Differences in IL-1Ra production in response to MDP in the eye versus plasma may influence, in part, the contribution of IL-1β in ocular and systemic inflammation. This report reveals a new function of NOD2 in vivo in the eye. In addition, our observations distinguish inflammatory effects of NOD2 in the eye from those of systemic NALP3 or NOD2 activation.


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MATERIALS AND METHODS
 
Reagents
Synthetic MDP (Bachem, Torrance, CA, USA) or LPS (Sigma Chemical Co., St. Louis, MO, USA) were dissolved in sterile saline. MDP tested below the lower limit of detection for endotoxin activity by Limulus amoebocyte lysate assay.

Mice
Age-matched (8–10 weeks) female BALB/c mice, IL-1R1 knockout (KO) mice, caspsae-1 KO mice, and their congenic (nonobese diabetic) controls were obtained from Jackson Laboratories (Bar Harbor, ME, USA). NOD2 KO mice were kindly provided by Dr. Richard Flavell (Yale University, New Haven, CT, USA). These mice were then backcrossed 10 generations onto a BALB/c background. Mice were housed in a facility approved by the Association of Assessment and Accreditation of Laboratory Animal Care International. Procedures were carried out according to National Institutes of Health (Bethesda, MD, USA) and Oregon Health and Science University Institutional Animal Care and Use Committee (Portland, OR, USA).

Intravital microscopy of the eye
For all imaging experiments, mice were given an intravitreal injection (2 µl) of 100 µg MDP or 250 ng LPS using a Hamilton syringe with a 301/2-gauge needle. After 5 h following treatment, the leukocytic response within the vasculature and extravascular tissue of the iris was assessed by intravital microscopy, according to a previously established method [24 ]. Briefly, at the time of imaging, animals were injected with 35 mg/kg rhodamine 6G (Sigma Chemical Co.), a fluorescent dye taken up by all leukocytes. Mice were anesthetized with 1.7% isofluorane, and the digital images of the iris vasculature were captured with a black and white video camera (Kappa Scientific, Gleichen, Germany) on an epifluorescence intravital microscope (modified Orthoplan; Leica, Wetzlar, Germany) in three independent regions of the eye. Diameter and length of each vessel segment or iris tissue as well as leukocyte phenotype (i.e., rolling, adhering, infiltrating) were quantified off-line with Image J analysis software and have been described in detail previously [24 ].

In vivo peritonitis model
Mice were administered an i.p. injection of 100 µg MDP in 0.5 ml sterile saline. After 5 h post-treatment, mice were killed by CO2 inhalation, and peritoneal lavage was performed using 7 ml PBS. The neutrophil response was quantified by flow cytometry using the neutrophil-specific marker Ly-6G-conjugated to PE and its PE-conjugated isotype control (BD PharMingen, San Jose, CA, USA). At the same time, blood was collected into heparin, and plasma levels of IL-1β and IL-1Ra were quantified by ELISA.

ELISAs
Protein was extracted from eye tissue, performed as described previously [25 ] with some modifications. Briefly, enucleated eyes were lysed in a buffer containing a protease inhibitor cocktail (Roche, Mannheim, Germany). Protein concentrations were determined using a bicinchoninic acid kit (Pierce-Endogen, Rockford, IL, USA), and equal amounts of protein for each sample were measured for IL-1β, IL-18, or IL-1Ra using commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA). Cytokine amounts were calculated as pg per µg sample protein in the eye tissue homogenates or represented as pg/ml for plasma levels.

Statistics
Data are represented as mean ± SEM. Mean differences were analyzed using two-way and one-way ANOVA with Bonferroni’s test or t-test post-hoc analyses. Differences were considered statistically significant when P < 0.05.


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RESULTS
 
Activation of NOD2 by MDP results in increased IL-1β and IL-18 in the mouse eye
As a result of the functional and structural similarities of NALP3 and NOD2, we hypothesized that activated NOD2 might function to promote IL-1β release in a caspase-1-dependent manner. We further postulated that IL-1β is a mediator of NOD2-induced inflammation. As the eye is one of the three tissues most consistently affected in Blau syndrome, the function of NOD2 within this specific tissue is critical to our understanding of the disease. Consequently, we sought to investigate whether activation of NOD2 by MDP results in IL-1β production within the eye via caspase-1. The present study used a quantity of MDP, a route of injection, and time-points that were optimized previously to demonstrate ocular inflammation [22 ].

We first tested whether activation of NOD2 increases IL-1β levels within the eye. Mice were treated with MDP or saline, and cytokine production was assessed over time (Fig. 1A ). We found that mice had significantly more IL-1β in their eyes within 3 h after MDP injection, and the level of IL-1 remained elevated out to 24 h following treatment. We also found that MDP resulted in a significant increase in IL-18 (Fig. 1B) , another cytokine regulated by the inflammasome.


Figure 1
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  		Figure 1. Activation of NOD2 by MDP results in increased IL-1β and IL-18 in the mouse eye. (A) BALB/c mice were treated with an intravitreal injection of 100 µg MDP or saline. Cytokine production of IL-1β in the eye was measured by ELISA as a function of time following treatment (left panel) or in NOD2 KO mice (right panel) at 6 h following treatment. (B) BALB/c mice were treated with an intravitreal injection of 100 µg MDP or saline. Cytokine production of IL-18 in the eye was measured by ELISA as a function of time following treatment (left panel) or in NOD2 KO mice (right panel) at 3 h following treatment. (C) NOD2 KO mice or BALB/c controls were treated with an intravitreal injection of 250 ng LPS or saline. Cytokine production of IL-1β was measured by ELISA at 3 h and 24 h following treatment. Data are the mean ± SEM; *, P < 0.05, comparison between treatments within a genotype; {dagger}, P < 0.05, comparison between treated KO and wild-type (WT) mice (n=6–10 mice/treatment/time).

We used NOD2 KO mice to test the requirement for NOD2 in these cytokine responses (Fig. 1) . We found that NOD2 KO mice did not show increased IL-1β or IL-18 production after MDP treatment (Fig. 1A and 1B , respectively), indicating that NOD2 plays a required role in their regulation within the eye. However, NOD2 KO mice are capable of generating IL-1β to other stimuli, as NOD2 KO mice treated with LPS show a significant increase in IL-1β levels within the eye to the same extent as WT controls (Fig. 1C) .

Caspase-1 is essential for IL-1β and IL-18 production in the eye in response to MDP
Caspase-1 is a crucial component of the inflammasome, as it is responsible for the proteolytic processing of IL-1β and IL-18 from its intracellular proforms to their active, secreted forms. These studies prompted us to further define the functional role of caspase-1 in the production of IL-1β and IL-18 in the eye in response to MDP. Caspase-1 KO mice and their controls were treated with MDP or saline, and IL-1β and IL-18 were measured (Fig. 2 ). We found that levels of IL-1β and IL-18 within MDP-injected eyes were reduced significantly in the absence of caspase-1. These data indicate that NOD2 activation results in production of IL-1β and IL-18 in a caspase-1-dependent manner in vivo.


Figure 2
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  		Figure 2. Caspase-1 is essential for MDP-induced production of IL-1β and IL-18. Caspase-1 KO mice and their controls (WT) were treated with an intravitreal injection of 100 µg MDP or saline. Cytokine production in the eye was measured by ELISA at 6 h for IL-1β (left panel) and at 3 h for IL-18 (right panel), the peak times for each cytokine response following treatment. Data are the mean ± SEM; *, P < 0.05, comparison between treatments within a genotype; {dagger}, P < 0.05, comparison between MDP-treated KO and WT mice (n=8–10 mice/treatment/genotype).

MDP-induced ocular inflammation occurs independently of IL-1β
To determine the role of caspase-1 in MDP-induced ocular inflammation, we used intravital microscopy to assess the ocular inflammatory response in mice deficient in caspase-1. The microscopy allows direct visualization of hallmarks of inflammation including leukocyte rolling and leukocyte intravascular adherence within the vasculature of the eye. Based on our previous studies, we have determined that inflammation in the eye is most robust at 6 h post-MDP treatment. Importantly, we have demonstrated that NOD2 KO mice failed to show intravascular inflammation within the eye in response to MDP, indicating an essential role for NOD2 in MDP-induced ocular inflammation [22 ]. Thus, we assessed differences in caspase-1 KO mice and their controls treated with MDP at this time (Fig. 3 ). To our surprise, we found that caspase-1 KO mice developed a significant intraocular inflammatory response to MDP with no significant difference between the caspase-1 KO mice and their WT controls. We also did not observe a difference in cellular infiltration in response to MDP in the caspase-1 KO mice (data not shown). As IL-18 production in the eye in response to MDP requires caspase-1, this finding further suggests that IL-18, like IL-1β, is not a likely mediator of ocular inflammation induced by MDP.


Figure 3
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  		Figure 3. MDP-induced ocular inflammation occurs independently of caspase-1, and caspase-1 KO mice and their controls (WT) were treated with an intravitreal injection of 100 µg MDP or saline. The ocular inflammatory response was assessed by intravital microscopy at 6 h following treatment (peak of inflammatory response to MDP). Two hallmarks of ocular inflammation, rolling leukocytes (left panel) and adhering leukocytes (right panel), were quantified. Data are the mean ± SEM; *, P < 0.05, comparison between treatments within a genotype; {dagger}, P < 0.05, comparison between MDP-treated KO and WT mice (n=8–10 mice/treatment/genotype/time).

We further assessed the IL-1β dependence of MDP-induced ocular inflammation in IL-1R1-deficient mice. IL-1R1 KO mice and their WT controls were treated with MDP or saline, and the ocular inflammatory response was assessed by intravital microscopy 6 h later (Fig. 4 ). Consistent with our findings in the caspase-1 KO mice, deficiency in IL-1R1 did not alter MDP-induced leukocyte rolling and adherence. We also did not observe a difference in cellular infiltration in response to MDP in the IL-1R1 KO mice (data not shown). Deficiency in IL-1R1 does not appear to alter the kinetics of MDP-induced ocular inflammation, as we have observed no significant alteration in inflammation at a later time-point, 24 h following treatment (data not shown).


Figure 4
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  		Figure 4. MDP-induced ocular inflammation occurs independently of IL-1β. IL-1R1 KO mice and their controls (WT) were treated with an intravitreal injection of 100 µg MDP or saline. The ocular inflammatory response was assessed by intravital microscopy at 6 h following treatment (peak of inflammatory response to MDP). Two hallmarks of ocular inflammation, rolling leukocytes (left panel) and adhering leukocytes (right panel), were quantified. Data are the mean ± SEM; *, P < 0.05, comparison between treatments within a genotype; {dagger}, P < 0.05, comparison between MDP-treated KO and WT mice (n=8–10 mice/treatment/genotype/time).

MDP-induced peritonitis is mediated, in part, by IL-1β and caspase-1
That MDP treatment elicited IL-1β production in the eye without an apparent contribution to the ocular inflammation prompted us to further question if the eye’s insensitivity to IL-1β may be unique. Accordingly, we sought to test the role of caspase-1 in the inflammatory response triggered by NOD2 activation using an in vivo model of MDP-induced peritonitis (Fig. 5 ). We found that i.p. injection of MDP resulted in a significant increase in plasma levels of IL-1β within 5 h of treatment, which was abolished in the absence of NOD2 (Fig. 5A) . We also tested the functional role of caspase-1 in the production of IL-1β in the plasma in response to systemically administered MDP and found that plasma levels of IL-1β were reduced significantly in the absence of caspase-1. However, the caspase-1 KO mice still demonstrated a partial ability to produce IL-1β compared with saline-treated KO mice.


Figure 5
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  		Figure 5. MDP-induced peritonitis is mediated, in part, by IL-1β and caspase-1. NOD2 KO, caspase-1 KO, and their corresponding WT controls were treated with an i.p. injection of 100 µg MDP. At 5 h following treatment, (A) plasma levels of IL-1β were measured by ELISA, and (B–D) neutrophil influx in peritoneal lavage fluid was quantified by flow cytometry. (B) A representative flow cytometry dot-plot depicting the presence of a neutrophil population in response to MDP. Data are the mean ± SEM; *, P < 0.05, comparison between treatments within a genotype; {dagger}, P < 0.05, comparison between treated KO and WT mice (n=8 mice/treatment/time). SSCH, ; FL2-H, fluorescence 2-height.

The involvement of NOD2 and caspase-1 in the recruitment of neutrophils in response to MDP treatment was examined. i.p. MDP treatment elicited a significant influx in neutrophils in peritoneal lavage fluid, as assessed by flow cytometry (representative dot-plot shown in Fig. 5B ). Importantly, NOD2 was required for neutrophil responsiveness to MDP, as the neutrophil influx was abolished in NOD2 KO mice (Fig. 5C) . In contrast to the eye, we observed a significantly diminished neutrophil response to MDP treatment in caspase-1-deficient mice compared with MDP-treated WT controls. However, the caspase-1 KO mice were still able to respond in part to MDP, as the neutrophil response was not abolished completely. These findings would indicate that in contrast to the eye, caspase-1 may contribute in part to peritonitis in this model of MDP-induced inflammation.

Differential IL-1Ra levels may influence the role of IL-1β in ocular and systemic responses
Our finding that the contribution of IL-1β in NOD2-mediated inflammation differed i.p. compared with ocular inflammation prompted us to examine IL-1R production in response to MDP. The bioactivity of IL-1β is regulated, in a large part, by IL-1Ra, which prevents IL-1β from interacting with IL-1R1. We examined endogenous production of IL-1Ra in the eye in response to intravitreal injection of MDP (Fig. 6A ). IL-1Ra levels in the eye did not alter significantly in response to MDP within a 24-h time window following treatment. In response to systemically administered MDP, levels of IL-1Ra in the plasma were increased significantly compared with saline controls (Fig. 6B) . Intriguingly, we observed that in untreated and naive animals, the baseline levels of IL-1Ra were significantly greater in the eye compared with the plasma (Fig. 6C) . Thus, the inflammatory action of IL-1β may be down-regulated by the quantity of IL-1Ra normally present within the eye, as the IL-1Ra levels in the eye are 14-fold greater than that of IL-1β. This may explain, in part, why IL-1β does not appear to influence the severity of MDP-induced uveitis but does influence MDP-induced neutrophil influx.


Figure 6
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  		Figure 6. Differential IL-1Ra levels may influence the role of IL-1β in ocular and systemic responses. (A) BALB/c mice were treated with an intravitreal injection of 100 µg MDP or saline. Production of IL-1Ra in the eye was measured by ELISA as a function of time following treatment. (B) BALB/c mice were treated with an i.p. injection of 100 µg MDP or saline, and plasma levels of IL-1Ra were measured by ELISA. (C) Truncal blood and eyes were collected from naïve BALB/c mice, and equal amounts of protein were assessed for IL-1Ra by ELISA in each tissue type and expressed as amount of IL-1Ra per ug protein. Data are the mean ± SEM; *, P < 0.05, comparison between treatments within a genotype; {dagger}, P < 0.05, comparison between treated KO and WT mice (n=5–8 mice/treatment/time).


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DISCUSSION
 
Here, we investigated the role for NOD2 in promoting IL-1β-mediated ocular inflammation. As the eye is one of the three tissues most consistently affected in Blau syndrome, the function of NOD2 within this specific tissue is critical to our understanding of the uveitis. We demonstrated that treatment with the NOD2 agonist MDP resulted in increased IL-1β within the eye in a NOD2- and caspase-1-dependent manner. However, the ocular inflammatory response occurred in the absence of IL-1 signaling, as mice deficient in caspase-1 or IL-1R1 still showed increased intravascular leukocyte rolling and adherence in response to MDP. The failure of IL-1β to mediate MDP-induced ocular inflammation was unexpected and differed compared with the role for IL-1β systemically in an in vivo model of MDP-induced peritonitis, wherein caspase-1-deficient mice showed a significant reduction in the neutrophil influx. Our results indicate the involvement of an IL-1β/caspase-1-independent mechanism in MDP-induced ocular inflammation. This is in contrast to NOD2-dependent, systemic responses, wherein we observed a significant contribution of caspase-1 in MDP-induced peritonitis. In addition, we conclude that although NOD2 and NALP3 might have similar capacities to activate inflammasome-driven cytokines, they likely differ in their downstream events that contribute to the pathology of their respective autoinflammatory syndromes or even within different tissue sites. NALP3 and caspase-1 have been demonstrated to be essential mediators in monosodium urate crystal-induced IL-1β production. Mice deficient in IL-1R1 have been shown to have abolished neutrophil infiltration in urate crystal-induced peritonitis [11 ].

Blau syndrome is an autoinflammatory disease, which has been postulated to involve IL-1β based on the homology of NOD2 (the gene responsible for Blau syndrome) and NALP3 (the gene responsible for CAPS). Notably, our findings here in the mouse are consistent with preliminary observations that we have made in patients with Blau syndrome. We have found that PBMC from patients with Blau syndrome do not appear to overproduce IL-1β (unpublished data). This would be in contrast to PBMC of CAPS patients that do show excessive IL-1β production [6 , 7 ]. In addition, we have treated two patients suffering from Blau syndrome with anakinra, and they have not enjoyed the sustained, therapeutic benefits that the CAPS patients do. Another group reported a positive clinical response in a single patient with Blau syndrome that was treated with anakinra [26 ]. Interestingly, the authors noted that although an improvement in the inflammatory symptoms occurred upon treatment with anakinra, the ocular inflammation persisted. It is intriguing to speculate whether this clinical observation relates to our findings here in mice, where IL-1β does not contribute to ocular inflammation but does contribute, in part, to the systemic form of inflammation triggered by MDP. The disparity in sensitivities to IL-1β in the eye versus systemic responses may be influenced by differences in IL-1Ra production—a finding that would also support the effectiveness for anakinra treatment for systemic inflammation but not for ocular inflammation.

The finding that IL-1β does not mediate ocular inflammation induced by MDP was unexpected. It is possible that the eye’s sensitivity to the inflammatory effects of IL-1β might be low as a result of rather high baseline levels of IL-1Ra in the eye, which were over 14 times that of the IL-1β levels after induction by MDP. The negligible role for IL-1β in MDP-induced uveitis would also be consistent with prior reports about endotoxin-induced uveitis, wherein IL-1β was determined to play only a partial role in ocular inflammatory responses [27 28 29 30 ]. We have determined that mice are capable of overcoming the suppressive effects of IL-1Ra with local treatment of recombinant IL-1β (data not shown). The aqueous humor is known to contain biologically active levels of several potent immunosuppressive factors such as TGF-β and the neuropeptides {alpha} melanocyte-stimulating factor and somatostatin [31 32 33 ]. Our observations indicate that IL-1Ra could be an additional immunoregulatory substance active within the eye.

The IL-1 cytokine family is continually growing along with the newly identified caspase-5 (or mouse caspase-11) as a participant in the inflammasome. The capacity of NOD2 to activate a different inflammasome or other IL-1 family members that might contribute to inflammation are yet to be determined. It has been postulated that cells such as monocytes require an initial signal for the transcription of IL-1β and IL-18 at the level of NF-{kappa}B activation [34 ]. As such, LPS or cycloheximide has been included as a cotreatment for activation of inflammasomes such as the NALP3 inflammasome [35 , 36 ]. We did not include an additional stimulus in our experiments, suggesting that in vivo, the eye might regulate IL-1β differently from what occurs in vitro in macrophage cell lines, or perhaps the fact that NOD2 contains a caspase activation and recruitment domain and activates NF-{kappa}B directly by way of its interaction with receptor-interacting protein 2 [37 ] renders NOD2 able to initiate both signals (i.e., transcription and processing/release of IL-1β via the inflammasome) autonomously. Indeed, we have observed that intravitreal MDP treatment increases mRNA expression of IL-1β in the eye (unpublished report) and evidence of its capacity to trigger transcription of IL-1β. Our finding that caspase-1 KO mice showed reduced IL-1β production in response to MDP supports a secondary role for NOD2 in the processing and/or release of IL-1β, as in this situation, NOD2 is intact and able to trigger transcription of IL-1β mRNA. As submission of this manuscript, a group from the Netherlands demonstrated that NOD2 appears to be involved in the transcriptional and post-translational processing of IL-1β in vitro in mononuclear cells [38 ], a finding consistent with our observations in vivo.

NF-{kappa}B is a pivotal transcription factor for a plethora of inflammatory mediators such as cytokines, chemokines, and adhesion molecules that could contribute to NOD2-driven ocular inflammation. Few studies of the functional consequences of NOD2 activation on inflammatory cascades or adhesion molecules in the eye have been performed. Our report published previously demonstrated a role for L-selectin in MDP-induced rolling cells within the vasculature of the eye in response to MDP [22 ]. Interestingly, we have observed a role for the cytokine IFN-{gamma} in promoting NOD2 inflammation in the eye (unpublished data), indicating a role for other inflammatory mediators besides IL-1β. Future studies are under way to explore such mechanisms.

These studies are the first step toward understanding the functions of NOD2 that might contribute to autoinflammation in the eye as occurs in Blau syndrome. The insights that have been gained by the study of rare diseases associated with NALP3 mutations should encourage similar investigations into NOD2-dependent, autoinflammatory syndromes. An in vivo model is an invaluable tool in this process.


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ACKNOWLEDGEMENTS
 
This project was funded by National Eye Institute grants F32-EY017254, EY015137, EY013093, and EY006484, along with Research to Prevent Blindness awards granted to J. T. R., S. R. P., and the Casey Eye Institute. We especially thank Kelley Goodwin and Hari Sawkar for their technical contributions.

Received January 9, 2008; revised March 18, 2008; accepted April 11, 2008.


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H. L. Rosenzweig, K. T. Galster, S. R. Planck, and J. T. Rosenbaum
NOD1 Expression in the Eye and Functional Contribution to IL-1{beta}-Dependent Ocular Inflammation in Mice
Invest. Ophthalmol. Vis. Sci., April 1, 2009; 50(4): 1746 - 1753.
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H. L. Rosenzweig, T. Kawaguchi, T. M. Martin, S. R. Planck, M. P. Davey, and J. T. Rosenbaum
Nucleotide Oligomerization Domain-2 (NOD2)-Induced Uveitis: Dependence on IFN-{gamma}
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