Keratoconus patients exhibit a distinct ocular surface immune cell and inflammatory profile

D’Souza, Sharon; Nair, Archana Padmanabhan; Sahu, Ganesh Ram; Vaidya, Tanuja; Shetty, Rohit; Khamar, Pooja; Mullick, Ritika; Gupta, Sneha; Dickman, Mor M.; Nuijts, Rudy M.M.A.; Mohan, Rajiv R.; Ghosh, Arkasubhra; Sethu, Swaminathan

doi:10.1038/s41598-021-99805-9

Cited by 22 publications

(24 citation statements)

References 76 publications

(86 reference statements)

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“…57 , 58 It was shown that delayed-type hypersensitivity, a CD4 + T-cell–dependent immune response, and corneal transplant rejection in mice are highly correlated. 59 , 60 Whereas in keratoconus patients a significant increase of ɣδ-T cells on the ocular surface was shown, 61 the presence of CD4 + or CD8 + T cells was not yet reported. In addition, the recruitment of CD4 + or CD8 + T cells for the alkali burn and suture placement models was not yet reported.…”

Section: Discussionmentioning

confidence: 97%

Different Murine High-Risk Corneal Transplant Settings Vary Significantly in Their (Lymph)angiogenic and Inflammatory Cell Signatures

Zhang

Schönberg

Bassett

et al. 2022

Invest. Ophthalmol. Vis. Sci.

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Purpose Pathologic conditions in the cornea, such as transplant rejection or trauma, can lead to corneal neovascularization, creating a high-risk environment that may compromise subsequent transplantation. This study aimed to evaluate the impact of different types of corneal injury on hemangiogenesis (HA), lymphangiogenesis (LA) and immune cell pattern in the cornea. Methods We used five different corneal injury models, namely, incision injury, alkali burn, suture placement, and low-risk keratoplasty, as well as high-risk keratoplasty and naïve corneas as control. One week after incision and 2 weeks after all other different injuries, corneal HA and LA were quantified by morphometric analysis. In addition, immune cell patterns of the whole cornea and the recipient rim were analyzed by immunohistochemistry. Immune cells in the draining lymph nodes (dLNs) were quantified by flow cytometry. Results Different types of corneal injury caused significantly different HA and LA responses (both P < 0.0001). The infiltration of corneal macrophages, dendritic cells, neutrophils, major histocompatibility complex (MHC) II + cells, CD4 + T cells, and CD8 + T cells varied significantly in different high-risk settings (all P < 0.0001). Both the expression of MHC II on macrophages ( P = 0.0005) and the frequency of MHC II + dendritic cells ( P = 0.0014) in the draining lymph nodes were significantly different across the various high-risk scenarios. Conclusions Murine high-risk settings caused by different underlying pathologies vary significantly in their (lymph)angiogenic and inflammatory cell patterns. Therefore, anti(lymph)angiogenic or immunomodulatory strategies to prevent and/or treat immune responses after subsequent corneal transplantation may need to be customized according to their immune-vascular “signatures.”

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Section: Discussionmentioning

confidence: 97%

Different Murine High-Risk Corneal Transplant Settings Vary Significantly in Their (Lymph)angiogenic and Inflammatory Cell Signatures

Zhang

Schönberg

Bassett

et al. 2022

Invest. Ophthalmol. Vis. Sci.

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“… 27 , 35 – 37 The demonstration of a positive correlation between the proportion of ocular surface immune cells and their specific secreted factors in the tear fluid suggests a contribution of immune cells to the ocular surface inflammatory milieu. 19 Recent studies have described the status of ocular surface immune cell subset proportions in DED patients. 18 , 20 , 21 , 27 , 38 , 39 Specifically, a direct association between ocular surface discomfort and the proportion of immune cells such as corneal dendritic cells, total leukocytes, neutrophils, and CD4 T cells has been reported.…”

Section: Discussionmentioning

confidence: 99%

“… 18 It is important to note that patients with keratoconus do not commonly complain about ocular surface discomfort, but the proportion of neutrophils and T cells has not been found to be elevated. 19 Hence, ocular surface discomfort may be associated with a distinct immune profile.…”

Section: Discussionmentioning

confidence: 99%

Ocular Surface Immune Cell Profiles in Contact Lens–Induced Discomfort

Nair

Sethu

Nagaraj

et al. 2022

Trans. Vis. Sci. Tech.

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Purpose Contact lens–induced discomfort (CLD) remains a primary factor in discontinuation or prevention of contact lens wear. Thus, we investigated the role of ocular surface immune cells in subjects with CLD. Methods Habitual contact lens (CL) wearers with CLD ( n = 19; 38 eyes) and without CLD ( n = 21; 42 eyes) as determined by the Contact Lens Dry Eye Questionnaire-8 was included in a trial. Enrolled subjects used either of the two types of CL (designated as CL-A or CL-D). Ocular surface cells from the bulbar conjunctiva were obtained by impression cytology. The collected cells were phenotyped using fluorochrome-conjugated antibodies specific for leukocytes (CD45 + ), neutrophils (CD66b +,High,Low ), macrophages (CD163 + ), T cells (CD3 + CD4 + , CD3 + CD8 + ), natural killer (NK) cells (CD56 +, High, Low ), natural killer T (NKT) cells (CD3 + CD56 + ), and gamma delta T (γδT) cells (CD3 + γδTCR + ) by flow cytometry. Further, corneal dendritic cell density (cDCD) was also determined using in vivo confocal microscopy. Results Significantly higher proportions of CD45 + cells were observed in subjects with CLD compared to those without CLD. The percentages of CD66b Total,Low , CD163 + , pan T cells, CD4 + T cells, CD8 + T cells, CD56 Total,High,Low (NK) cells, and NKT cells, as well as the CD4/CD8 ratio, were significantly higher in CLD subjects. The proportion of T cells (CD4, CD8, CD4/CD8 ratio, NKT cells) and macrophages exhibited a direct association with discomfort score. The percentages of CD45 + , CD66b Total,Low , CD163 + , CD3 + , CD56 Total,High,Low , and NKT cells and cDCD were significantly higher in CLD subjects wearing CL-D. The percentages of CD66b High , CD4 + T cells, CD8 + T cells, NKT cells, and CD4/CD8 ratio were significantly higher in CLD subjects wearing CL-A. Conclusions Increased proportions of ocular surface immune cells are observed in CLD, and the lens type could impact the immune cells associated with CLD. Translational Relevance The association between the proportion of altered ocular surface immune cell subsets and contact lens–induced discomfort underpins the importance of considering immune-related aspects ...

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“…This will most likely give more promising results in vivo as severe downregulation of LOX is associated with KC. 220 Too little LOX expression will cause corneal thinning and will cause changes in light scattering resulting in astigmatism or blurry vision as seen in KC. 270 Immunofluorescence examination of LOX in rabbit eyes exposed to SM vapor in vivo showed a time-dependent increase in LOX protein expression.…”

Section: Discussionmentioning

confidence: 99%

“…This will most likely give more promising results as severe downregulation of LOX is associated with keratoconus. 220 Upstream markers for the LOX include p38 and PI3K.…”

Section: Why Study Lysyl Oxidase? An Un-explored Role Of Lox In Corne...mentioning

confidence: 99%

Mechanistic understanding and potential targets for mustard gas keratopathy

Sinha¹

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Sulfur mustard gas (SM), a vesicating and alkylating agent, has been used as a warfare agent since World War I and recently in Syria. SM exposure causes severe corneal injury, pain, and irreversible blindness. The cornea provides twothirds of the eye's light refraction vital for vision. Clinically, SM rapidly penetrates the cornea after exposure and causes a grade-3 or grade-4 clinical pathology called mustard gas keratopathy (MGK). MGK is characterized by ocular inflammation, loss of epithelial barrier, recurrent epithelial erosions, epithelialstromal separation, haze/fibrosis, and neovascularization in the cornea. Despite the high risk of SM for terrorism and mass obliteration, the pathophysiological mechanisms associated with MGK remain elusive. MGK-related corneal haze/fibrosis results from the transdifferentiation of keratocytes into pathological cellular phenotype called myofibroblasts that cause massive overproduction of collagen during wound healing. Keratocytes and collagens residing in the stroma play an essential role in maintaining corneal transparency and are regulated by cytokines and chemokines. The predominant collagens in the stroma are Type I, V, and VII. Additionally, a unique arrangement of collagen fibrils is required for transparency and corneal function. Collagen fibrils are narrow with uniform diameter and arranged with a high degree of lateral ordering. The mechanisms involving myofibroblast formation and collagen derangement after mustard gas exposure are unknown. Presently, a major knowledge gap is how SM exposure affects myofibroblast generation and collagen fibril arrangement in the wounded cornea. My dissertation fills this knowledge gap through successful testing of a novel central hypothesis that mustard gas exposure to the eye results in corneal haze/fibrosis by excessive production of myofibroblasts via TGFβ/SMAD signaling and alters the distinctive organization of collagen fibrils by regulating LOX activity via PI3K/MAPK signaling in the stroma. The formation of haze/fibrosis post SM exposure was tested using an in vivo rabbit model, in which live New Zealand White rabbits received SM vapor exposure localized to the eye. The goals of my PhD thesis were to characterize time-dependent changes in the stroma in live animals in situ employing eye examinations and state-of-the-art multimodal clinical ophthalmic imaging techniques in live rabbits with Stereo, Slit-lamp, HRT-RCM3, and Spectralis microscopy system. Fantes grading, fluorescein staining, Schirmer's tests, pachymetry, and applanation tonometry were conducted to measure levels of corneal haze, ocular surface healing, tears, corneal thickness, and intraocular pressure, respectively. H and E and PSR staining were used for histopathological cellular changes in the cornea. In vivo confocal and OCT imaging revealed significant changes in structural and morphological appearance of the cornea in vivo in SM-exposed rabbit corneas in a time-dependent manner compared to naive cornea (p [less than] 0.001 or p [less than] 0.0001). Also, SM-exposed eyes showed loss of corneal transparency characterized by increased stromal thickness (p [less than] 0.0001), debris, and light-scattering myofibroblasts or activated keratocytes, representing haze formation in the cornea. Naive eyes did not show any structural, cellular, and functional abnormalities. SM exposure showed increased central corneal thickness, ocular edema and tearing compared to the eyes of the naive (p [less than] 0.001 or p [less than] 0.0001). This study concluded that structural and cellular changes in primary corneal layers are early pathological events contributing to MGK in vivo. Next, I tested if TGFβ/SMAD is a major molecular mechanism for myofibroblast formation following SM exposure. Rabbit corneas exposed to SM had a time-dependent increase in [alpha]SMA, a maker for myofibroblast formation. Additionally, using an established in vitro model of primary human corneal fibroblasts (hCSF) treated with nitrogen mustard (NM), an analogous agent to SM, or NM+SMAD inhibitors, I verified my hypothesis that SMAD is a predominant pathway for myofibroblast formation in MGK. Primary hCSF treated with NM showed a significant time-dependent increase (p [less than] 0.05, p [less than] 0.01) in [alpha]SMA expression which was significantly decreased by SMAD inhibitors (p [less than] 0.05, p [less than] 0.01). This data strongly supported my notion that SMAD is a major pathway for myofibroblast formation in corneal stroma after mustard gas exposure. The final section of my thesis tested another novel postulate that lysyl oxidase (LOX) is a possible mechanism to cause changes in the collagen lamella. LOX activity regulates extracellular cross-linking; however, literature/knowledge on LOX function in the cornea is very limited. Thus, I tested if p38 and PI3K are major regulatory pathways. p38 and PI3K have shown to regulate steady state LOX mRNA in cardiac fibroblasts. An in vitro study using hCSF cells, NM, p38 inhibitor, and PI3K inhibitor concluded that (a) p38 follow the pattern similar to SMAD signaling, and (b) PI3K was directly related to LOX production. Subsequent research showed that p38 has a function correlative to SMAD function in the corneal epithelium. This finding was the first to identify the role of PI3K in the mustard gas exposed corneal stroma. In conclusion, my dissertation identified time-dependent in situ structural and cellular aberrations in the rabbit cornea in vivo after mustard gas exposure and remarkably improved mechanistic knowledge that will be instrumental in developing novel strategies and molecular targets for newer therapeutics to prevent and treat corneal mustard gas keratopathy. Additionally, it might help understanding corneal pathologies caused by other alkylating/vesicating agents.

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Keratoconus patients exhibit a distinct ocular surface immune cell and inflammatory profile

Cited by 22 publications

References 76 publications

Different Murine High-Risk Corneal Transplant Settings Vary Significantly in Their (Lymph)angiogenic and Inflammatory Cell Signatures

Different Murine High-Risk Corneal Transplant Settings Vary Significantly in Their (Lymph)angiogenic and Inflammatory Cell Signatures

Ocular Surface Immune Cell Profiles in Contact Lens–Induced Discomfort

Mechanistic understanding and potential targets for mustard gas keratopathy

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