S. R. Planck

Trauma and alkali burns induce distinct patterns of cytokine gene expression in the rat cornea

Cytokines such as the interleukins (IL) and tumor necrosis factor alpha (TNF alpha) have traditionally been associated with paracrine regulation of immune reactions. These proteins also have properties suggestive of functional roles in the inflammatory and reparative responses to tissue injury. In this study, mRNA levels for IL-1 alpha, IL-1 beta, IL-6, TNF alpha, interferon gamma, transforming growth factor beta 1, and CD4 were monitored in rat corneas at times from 1 hour through 2 weeks after incisional trauma or alkali burns. Transcripts for IL-1 alpha, TNF alpha, and TGF beta 1 were present in most corneal samples; whereas those for IFN gamma and CD4 were not detected. As early as 1 hour following either of these non-immunologic forms of injury, expression of IL-6 mRNA levels was induced. Only in corneas with alkali burns did IL-6 induction persist from days 1 through 7. The alkali-injured corneas also had markedly increased IL-1 beta mRNA levels from days 1 through 7. These observations indicate that cytokine mRNA is induced in the cornea by trauma without an apparent immunologic stimulus. Our data are consistent with the hypothesis that corneal tissues respond to different types of injury with different patterns of cytokine gene expression.

Cloning, sequencing and expression analysis of the mouse NOD2/CARD15 gene

Abstract:Background: Mutations in the human NOD2/CARD15 gene have been associated with Crohns disease and Blau syndrome. The objective of the present study was to clone the murine form of NOD2 and characterize its tissue distribution, function and response to inflammatory stimuli. Methods: Murine NOD2 was isolated using anchored polymerize chain reaction (PCR). Sequence analysis confirmed the identification of full-length cDNA representing the murine NOD2 gene. Using this sequence to search a Mus musculus supercontig database, NOD2 genomic DNA was identified. NOD2 was transfected into human embryonic kidney (HEK) cells and nuclear factor kappa B (NF-κB) activation was measured using a reporter assay. Tissue distribution and changes in transcription in mouse monocytes in response to inflammatory stimuli was determined by real time PCR. Results: The NOD2 gene spans 39 KB and contains 12 coding exons on chromosome 8. Expression of mouse NOD2 into HEK cells resulted in NF-κB activation. NOD2 was found to be expressed in all mouse tissues analyzed except skin, with highest levels in lung, thymus and spleen. NOD2 mRNA levels increased greater than two-fold in a monocyte cell line in response to lipopolysaccharide, lipoteichoic acid, interferon-g and tumor necrosis factor-α. Conclusions: Common structural and functional features between human and mouse NOD2 were identified. This should allow for development of relevant animal models to evaluate the role of NOD2 in chronic inflammatory disorders.

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

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.

Endotoxin-induced uveitis is primarily dependent on radiation-resistant cells and on MyD88 but not TRIF

TLR4 activation by LPS (endotoxin) is mediated by the MyD88 and TRIF intracellular signaling pathways. We determined the relative activation of these pathways in murine ocular tissue after LPS exposure. Additionally, we explored whether BM‐derived or non‐BM‐derived cells were the major contributors to EIU. Mice deficient in TRIF or MyD88 and their congenic (WT) controls received 250 ng ultrapure LPS ivt at 0 h. Ocular inflammation was assessed by histological analysis at 4, 6, and 24 h, and additionally, in MyD88−/− mice, intravital microscopy was performed at 4 h and 6 h to assess adherent, rolling, and infiltrating cells in the iris vasculature and tissue. Cytokines associated with the MyD88 and TRIF intracellular signaling pathways were analyzed in ocular tissue at 4 h. BM chimeric mice (WT→WT, TLR4−/−→WT, WT→TLR4−/−) received 250 ng LPS by ivt injection, and ocular tissues were examined by histology at 6 h. Lack of MyD88 resulted in a markedly diminished cellular response and reduced production of MyD88‐related cytokines 4 h post‐LPS treatment. In contrast, lack of TRIF led to reduced production of TRIF‐related cytokines and no change in the cellular response to LPS. Therefore, the MyD88 pathway appears to be the dominant TLR4 pathway in EIU. Only WT → TLR4−/− chimeric mice were resistant to EIU, and this suggests, surprisingly, that non‐BM‐derived (radiation‐resistant) cells in the eye play a greater role than BM‐derived cells.

Activation of nucleotide oligomerization domain 2 exacerbates a murine model of proteoglycan-induced arthritis

In addition to its role in innate immunity, nucleotide oligomerization domain 2 (NOD2) has been shown to play a suppressive role in models of colitis. Notably, mutations in NOD2 cause the inherited granulomatous disease of the joints called Blau syndrome, thereby linking NOD2 with joint disease as well. However, the role of NOD2 in joint inflammation has not been clarified. We demonstrate here that NOD2 is functional within the mouse joint and promotes inflammation, as locally or systemically administered muramyl dipeptide (MDP; the NOD2 agonist) resulted in significant joint inflammation that was abolished in NOD2‐deficient mice. We then sought to investigate the role of NOD2 in a mouse model of inflammatory arthritis dependent on adaptive immunity using TCR‐transgenic mice whose T cells recognized the dominant epitope of proteoglycan (PG). Mice immunized with PG in the presence of MDP developed a more severe inflammatory arthritis and histopathology within the joints. Antigen‐specific activation of splenocytes was enhanced by MDP with respect to IFN‐γ production, which would be consistent with the Th1‐mediated disease in vivo. Intriguingly, NOD2 deficiency did not alter the PG‐induced arthritis, indicating that NOD2 does not play an essential role in this model of joint disease when it is not activated by MDP. In conclusion, we demonstrate that in a model of inflammatory arthritis dependent on T and B cell priming, NOD2 activation potentiates disease. However, the absence of NOD2 does not alter the course of inflammatory arthritis, in contrast to models of intestinal inflammation.

Anterior uveitis accompanies joint disease in a murine model resembling ankylosing spondylitis.

Background: Uveitis is often associated with a systemic inflammatory disease such as ankylosing spondylitis. Our understanding of the eye’s susceptibility to immune-mediated uveitis as in the apparent absence of infection has been limited by a relative lack of experimental models. Here we sought to assess whether ocular inflammation occurs in a previously described murine model of proteoglycan-induced spondylitis, wherein mice develop progressive spondylitis, sacroiliitis and peripheral arthritis – features common to the clinical presentations of ankylosing spondylitis. Methods: Using intravital microscopy we examined the ocular inflammatory response after the onset of arthritis in mice that overexpressed the T cell receptor (TCR) specific for a dominant arthritogenic epitope of cartilage proteoglycan [TCR-Tg (transgenic) mice] or BALB/c controls. Results: Immunized TCR-Tg mice showed a significant increase in the number of rolling and adhering cells within the iris vasculature compared to adjuvant control mice. Cellular infiltration within the iris tissue, as assessed by intravital microscopy and histology, was also increased. Our initial temporal analysis has revealed that immunized TCR-Tg mice show a significant increase in intravascular inflammation by 2 weeks after immunization, but it diminishes at 4 weeks after immunization. Conclusions: Although these data are preliminary, this model has the potential to clarify the mechanisms accounting for the coexistence of eye and sacroiliac inflammation as occurs in patients with ankylosing spondylitis.