In Vivo Corneal Confocal Microscopy and Histopathology of Keratitis Fugax Hereditaria from a Pathogenic Variant in NLRP3

Purpose: To apply in vivo corneal confocal microscopy (IVCM) to study the pathogenesis of keratitis (keratoendotheliitis) fugax hereditaria, an autosomal dominant cryopyrin-associated periodic keratitis, associated with the c.61G>C pathogenic variant in the NLRP3 gene, in its acute and chronic phase, and to report histopathological findings after penetrating keratoplasty. Design: Observational case series Methods: Study population: Six patients during an acute attack, 18 patients in the chronic phase, and one patient who underwent penetrating keratoplasty. Intervention: Sanger sequencing for the NLRP3 variant c.61C>G. Clinical examination, corneal photography, IVCM, light microscopy and immunohistochemistry. Main Outcome Measures: IVCM and histopathological findings. Results: During the acute attack, hyperreflective cellular structures consistent with inflammatory cells transiently occupied the anterior to middle layers of the corneal stroma. Other corneal layers were unremarkable. With recurring attacks, central oval stromal opacities accumulated. IVCM revealed that they contained long hyperreflective needle-shaped structures in extracellular matrix. By light microscopy, the anterior half of the stroma displayed thin and finely vacuolated lamellae, and keratocytes throughout the stroma were immunopositive for syndecan. Conclusions: The acute attacks and chronic stromal deposits mainly involve the anterior to middle layers of the corneal stroma, and the disease is primarily a keratitis rather than a keratoendotheliitis. IVCM shows that inflammatory cells invade only the stroma during an acute attack. IVCM and light microscopic findings suggest that the central corneal opacities represent gradual deposition of extracellular lipids. The disease could make a good in vivo model to study activation of the NLRP3 inflammasome in cryopyrin-associated periodic syndromes. Turunen et al. In vivo Corneal Confocal Microscopy of Keratitis Fugax Her editaria 1 In Vivo Corneal Confocal Microscopy and Histopathology of Keratitis Fugax Hereditaria from a Pathogenic Variant in NLRP3 Joni A. Turunen, 1,2* Annamari T. Immonen, 1 Reetta-Stiina Järvinen, 1,2 Sabita Kawan, 1,2 Pauliina Repo, 1,2 Anna Korsbäck, 1 Olli Ala-Fossi, Aino M. Jaakkola, 1 Anna Majander, 1 Minna Vesaluoma, 1 Tero T. Kivelä Department of Ophthalmology, Helsinki University Ho spital and University of Helsinki, Helsinki, Finland Folkhälsan Research Center, Biomedicum Helsinki, He lsinki, Finland *Corresponding Author and Address for Reprints: Jon i A. Turunen, Department of Ophthalmology, Helsinki University Hospital, Haartm aninkatu 4 C, PL220, FI-00029 HUS, Helsinki, Finland. Email: joni.turunen@helsinki.fi, tel: +358 50 544 9 088, fax +358 9 4717 5100 Short title: In vivo Confocal Microscopy of Keratitis Fugax Hereditaria


Introduction
Keratoendotheliitis fugax hereditaria (MIM 148200), initially called keratitis fugax hereditaria, is a cryopyrin-associated periodic keratitis that predominantly, if not exclusively, affects the cornea. [1][2][3] It is inherited in an autosomal dominant pattern and is emerging as relatively frequent in Finland. 3 The episodic attacks begin at the median age of 11 years and recur 1 to 6 times per year. The attack is characterized by unilateral pain, pericorneal injection, and photophobia. It usually lasts 1 to 2 days, but vision can remain blurry for several weeks. The attacks become milder and decrease in frequency in middle age. Repeated episodes result in bilateral horizontally oval central stromal opacities in approximately half of the patients, some of whom experience permanently reduced vision. The patients have no systemic symptoms or signs during the acute episodes. 3 We recently discovered that keratitis fugax hereditaria is associated with a heterozygous pathogenic variant c.61G>C in the nucleotide-binding domain, leucine-rich repeat family, pyrin domain-containing 3 (NLRP3) gene. 3 The NLRP3 protein is expressed mainly by macrophages, but also in corneal tissue. 4,5 This variant corresponds to the amino acid substitution p.Asp21His in NLRP3, also known as cryopyrin, a component of the inflammasome. The inflammasome is a large multiprotein complex that plays a crucial role in innate immunity, and can be activated by various stimuli, including microbes. Other known mutations in NLRP3 cause three monogenic, autoinflammatory, cryopyrin-associated periodic syndromes (CAPS): familial cold autoinflammatory syndrome (FCAS1, MIM 120100), Muckle-Wells syndrome (MWS, MIM 191900), and chronic infantile neurological cutaneous articular syndrome (CINCA, MIM 607115) also known as neonatal-onset multisystem inflammatory disease. 6 The three syndromes share corneal phenotypes with keratitis fugax hereditaria. 7,8 During acute attacks, a pseudoguttata-like corneal phenotype was observed with specular microscopy, 2,3 leading to the term keratoendotheliitis, but all patients had normal endothelial cells between attacks. 3 In some patients, an anterior chamber reaction was observed. After several episodes, corneal stromal opacities frequently evolve. The corneal thickness is slightly increased during acute attacks, but after opacities have developed, the thickness will eventually decrease. 3 To elucidate the nature and pathogenesis of the acute episodes and of the persistent corneal opacities in keratitis fugax hereditaria, we performed in vivo corneal confocal microscopy (IVCM) in six patients during an acute attack and in twelve patients, some of whom had typical horizontally oval corneal stromal opacities, during the quiet phase. Moreover, we identified one patient carrying the pathogenic variant who in the past had undergone bilateral penetrating keratopathy because of typical chronic stromal opacities.

Patients
Patients who had genetically confirmed keratitis fugax hereditaria were eligible to this observational cohort study that was approved by the institutional review board of the Operative Section of the Helsinki University Hospital and followed the tenets of the Declaration of Helsinki. We obtained written informed consent from all participants. Additionally, one of us scanned the records of 2197 surgeries of 1673 patients who had undergone any type of keratoplasty between January 1, 1995, and December 31, 2015, and had the removed corneal disk archived in the Ophthalmic Pathology Laboratory, Helsinki University Hospital, Finland, for corneal opacities consistent with keratitis fugax hereditaria. We identified one female patient with preoperative anterior segment photographs that were consistent with this diagnosis and who had her left eye operated in 1998 and the right eye in 2004.

Genetic analysis
The NLRP3 variant c.61G>C (rs200154873; GenBank: NM_004895.4) was confirmed by Sanger sequencing in all participants, including the patient who had undergone penetrating keratoplasty in the past, as described earlier. 3

Clinical examination
We performed a comprehensive ophthalmic examination of eighteen patients. Standardization of Uveitis Nomenclature (SUN) Working Group definition were used to grade anterior chamber cells. 9 We also performed anterior segment photography, non-contact specular microscopy (EM-3000; Tomey, Nagoya, Japan), and Fourier-domain, swept source anterior segment optical coherence tomography (SS-1000 CASIA; Tomey).

In vivo confocal microscopy
After instilling a drop of 1% tetracaine hydrochloride (Minims Tetracaine Hydro 1%, Bausch & Lomb) to the lower conjunctival sac, corneal images were obtained with the Heidelberg Retina Tomograph 3 equipped with the Rostock Cornea Module (HRT III RCM; both from Heidelberg Engineering GmbH, Dossenheim, Germany) according the manufacturer's instructions. We imaged the central cornea along the sagittal axis so that we successively captured the epithelium, subepithelial neural plexus, anterior stroma, posterior stroma, and endothelium.

Results
Of the eighteen patients imaged, seven were described in our previous genetic study. 3 Here were add five new families, three of which had several affected members (Supplementary Table 1, Turunen et al.

Acute attack
We imaged six patients within 1 to 2 days after the onset of symptoms during an acute attack of keratitis. All of them reported typical symptoms: a unilateral foreign body sensation that transformed into pain, followed by blurry vision. None reported systemic symptoms. Clinically, reduced visual acuity, conjunctival injection, and corneal haze were present. Most patients had cells in the anterior chamber, corresponding to SUN 0.5+, but patient 15-03 had SUN 2+ anterior uveitis. Specular microscopy showed cornea pseudoguttata as reported earlier. 2,3 During the attack, corneal thickness was slightly increased (45 µm) as compared with the fellow eye, as measured using corneal tomography. IVCM showed a normal epithelium with some hyperreflective dendritiform elements at the level of the basal epithelial cells and Bowman's layer ( Fig.1A and B). We imaged numerous hyperreflective small roundish bodies 10-15 µm in diameter in the middle stromal layers of the central cornea, most probably representing infiltrating inflammatory cells ( Fig. 1C and D). The posterior stroma and endothelium appeared to be essentially normal although subtle endothelial gaps and blebs were seen ( Fig.1E and F). We also documented identical migration to the middle stromal layers of putative inflammatory cells in varying numbers in all six patients from five families that we imaged (5-02, 5-04, 7-05, 15-03, 16-01 and 20-01) during their acute attack ( Fig.  1G-L). The cells were often arranged in clusters or rows. Most keratocyte nuclei appeared relatively normal, although some were hyperreflective. In some corneae isolated needle-shaped bodies were found (Fig. 1J). We documented similar cells in Pseudomonas keratitis, supporting their inflammatory nature (Supplemental Fig. 2).

Chronic phase
We imaged eighteen patients with IVCM in the chronic phase, including the six patients imaged during an acute attack. Again, the epithelium and subepithelial nerves were normal with hyperreflective dendritiform elements at the level of Bowman's layer ( Fig. 3A and B). Coinciding with the central horizontally oval opacity, we imaged 50-200 µm long highly reflective, strictly linear, needle-shaped elements toward the middle stromal layers (Fig. 3C-E). The nuclei of keratocytes appeared to be relatively normal. The endothelial cells could display polymegethism (Fig.3F). Similar needle-shaped structures were present in some patients with clinically clear corneas, but to a lesser degree. Three patients had only a few of these structures (patients 3-03, 16-01 and 16-04). Altogether, we detected at least some of them in most (67%) patients ( Fig. 3G-L).

Ophthalmic pathology
After four decades of typical symptoms and consequent development of typical bilateral central horizontally oval corneal stromal opacities, a female patient underwent penetrating keratoplasty of her left and right eye at the age of 48 and 53 years, respectively. We have not encountered any other patient who would have had corneal surgery. During the preceding decade, her opacities had remained essentially stationary (Fig. 4A and B). The removed corneal disks showed a normal epithelium and an intact 12 µm thick Bowman's layer with solitary leukocytes under the basal epithelial cell layer (Fig. 4C and D). The stroma was thin, leading to central corneal thickness of 320 µm and 270 µm in the right and left eye, respectively. Corresponding to the central opacity, the anterior half of the stromal lamellae were thinner than those in the anterior peripheral part of the disk (Fig. 4C) and in the deeper central stroma (Fig. 4D) and finely vacuolated, whereas only minor vacuoles could be discerned in the peripheral and deep stromal lamellae that also were relatively thin. Descemet's membrane was 3 µm thick and thin for age. We counted on average 38 nuclei of endothelial cells per mm, which is typical for age.
Periodic acid-Schiff, Masson's trichrome and Congo red stain revealed no stromal deposits. Oil red O staining was technically inadequate as frozen sections were not available. Immunostaining of stromal keratocytes for vimentin and the CD34 epitope ( Fig. 4E and F) was somewhat uneven compared with that typically seen in normal cornea. The keratocytes were also immunolabeled for syndecan-1, and the reaction was more pronounced and granular in the anterior central stroma with thin vacuolated lamellae (Fig. 4G). We detected no α-smooth muscle actin-immunopositive myofibroblasts or CD163-immunopositive macrophages (Fig. 4H). No polymorphonuclear leukocytes were present. A few scattered T-cells were found both within and beneath the epithelium and among the anterior most stromal lamellae reacted with antibodies against CD3, CD4 and CD8 (Fig. 4I-K).

Discussion
In this first IVCM study of the cryopyrin-associated periodic corneal inflammation known as keratitis or keratoendotheliitis fugax hereditaria, six patients who carried the variant c.61G>C in NLRP3 that leads to Asp21His substitution in cryopyrin showed during their acute attack invasion of the corneal stroma by hyperreflective structures that we interpreted as inflammatory cells. Detection of inflammatory cells using IVCM is based on the morphology and size of such hyperreflective structures. 12 Cryopyrin is a crucial component of the NLRP3 inflammasome that, when activated, leads to release of interleukin-1β. The symptoms and signs, the transient corneal haze, and the accompanying anterior chamber reaction support the identification of the hyperreflective structures as inflammatory cells. This interpretation is further supported by a recent study in which experimental activation of the NLRP3 inflammasome recruited neutrophils to the mouse cornea, a reaction that was markedly weaker in NLRP3 knockout mice. Such cells in our patients were absent between attacks, and we detected only a few scattered T-lymphocytes and no polymorphonuclear leukocytes in two penetrating keratoplasty specimens from one patient between attacks.
In corneal autoimmune diseases, inflammatory cells are usually located in the limbal region or in the peripheral cornea, whereas in keratitis fugax hereditaria, an autoinflammatory disease, we observed putative inflammatory cells from the periphery to the central stroma. Currently, it is unknown what triggers the inflammatory reaction and homes the cells to the corneal stroma, and how they disappear after the attack. In a rabbit model of corneal injury, inflammatory cells also disappear almost completely in 24 hours after injury. 13 Classification of inflammatory cells is not possible using IVCM either. 14 The NLRP3 protein is mainly expressed in monocytes and macrophages, 15 which we did not detect in the penetrating keratoplasty specimens in the quiet phase. It has also been reported that corneal cells express cryopyrin. 5 In contrast to the pseudoguttata appearance of the corneal endothelium as has been observed by specular microscopy 2,3 , IVCM documented uninvolved corneal endothelium during acute attacks. The pseudoguttata-like phenotype is most probably the result of a masking effect caused by the stromal clusters of inflammatory cells. The term keratoendotheliitis 2,3 thus might be misleading, and we recommend reverting to the initial description keratitis fugax hereditaria 1 like we have done in the present report. While we cannot exclude that secondary endotheliitis might be present in some patients, the endothelium is not the predominant site of inflammation.
After repeated attacks, central stromal opacities develop as was seen in twelve of the eighteen c.61G>C variant carriers imaged between attacks. These faint to distinct, slowly progressive opacities reduce light transmittance, and visual acuity decreases accordingly. 3 The opacities bear resemblance to the disk-like central opacities in Schnyder corneal dystrophy without crystals. 16 The fine vacuolization of the anterior half of the central corneal stroma is seen both in Schnyder corneal dystrophy and in lipid keratopathy from various causes, and would be consistent with deposition of intrastromal lipid in keratitis fugax hereditaria. Finally, the needleshaped structures found in the extracellular matrix of the middle stromal layers by ICVM closely resemble those described in some patients with Schnyder corneal dystrophy [17][18][19][20] .
Using IVCM, very similar needle-shaped structures have been described in several corneal diseases without lipid deposition as well, including microbial keratitis, 21 Mooren's ulcer, 12 and chemical injury, 22 and postoperatively after penetrating keratoplasty, 23 Descemet stripping automated endothelial keratoplasty, 24 femtosecond laser-assisted keratoplasty, 25 corneal crosslinking, 26 and wound repair after LASIK. 27 In these contexts, needle-shaped elements have generally been interpreted as activated keratocytes. 28 Other authors have proposed that the needle-like structures might represent either lipofuscin 18 or disorganized extracellular material. 25 This theory would be supported by our observation that keratocytes in keratitis fugax hereditaria were prominently immunolabeled for syndecan-1, which is a sign of activation. Interestingly syndecan also has been implicated as a negative regulator of leukocyte-mediated inflammatory responses. 29 However, the studies mentioned mostly show single, faint and scattered needleshaped structures and not such dense aggregates that we found. Moreover, we found no evidence of a scarring reaction in the form of myofibroblasts and macrophage infiltration.
We favor the theory that in the chronic phase of keratitis fugax hereditaria, the needle-shaped elements might correspond to hyperreflective extracellular matrix around degenerated collagen fiber bundles associated with deposition of lipid. These changes would originate from repeated release of inflammatory cytokines and from waves of disintegrating leukocytes. Such degenerative changes would gradually lead to thinning of corneal lamellae, especially in the anterior stroma. This theory finds support from the unusual granular syndecan deposition in the anterior corneal stroma.
The cornea is transparent because of its highly specialized ultrastructure where the collagen lamellae have a parallel three-dimensional arrangement and uniform spacing between fibers. Interfibrillar spacing and proteoglycan content of the extracellular matrix is even more critical to corneal transparency. Stromal debris causes corneal haze and extracellular matrix backscatter is seen in IVCM. 30 Another theory is that stem cell-like keratocytes may have a role in structural adaptation by minimizing light scatter in the cornea. When keratocytes change from quiescent to activated ones, their biochemical homeostasis changes as well, and this increases light scatter. [31][32][33][34] The former process appears more likely in keratitis fugax hereditaria. However, it is unclear why the opacities develop only in the center of the cornea. Proximity to the limbal circulation might somehow offer protection or clearance.

In vivo Corneal Confocal Microscopy of Keratitis Fugax Hereditaria 7
Our data confirm that keratitis fugax hereditaria is a cryopyrin-associated periodic corneal autoinflammation mainly involving anterior to middle stromal layers. We also demonstrate that corneal endothelium is not notably affected, and thus the term keratoendotheliitis is not correct. After repeated attacks, corneal opacities develop that by ICCM consist of hyperreflective needleshaped structures and may reflect both degenerated stromal extracellular matrix and deposition of lipid. Currently, we do not know why the patients carrying the variant p.Asp21His in NLRP3 only have symptoms in the anterior segment of the eye, and why other tissues are not affected as they are in other cryopyrin-associated periodical syndromes from pathogenic variants in NLRP3. 7,8,35,36 Because of its transparency and avascularity, the cornea and keratitis fugax hereditaria could potentially make a good in vivo human model to study the activation, regulation and tissue specificity of the NLRP3 inflammasome.