FP1577 : Translating gel to science! – Vitreous Immuno-profiling

Dr.Thirumalesh M B, Dr.Rohit Shetty

Translating gel to science! – Vitreous Immuno-profiling 

Purpose:

To describe the immunological profile of cellular and soluble factors of the vitreous in patients with proliferative diabetic retinopathy compared to non-diseased vitreous (of macular hole patients).

Methods:

A descriptive study of 15 eyes of 15 patients who underwent vitrectomy for diabetic TRD/vitreous hemorrhage. Their vitreous was collected and immunological profiling was performed. This was compared with immune-profiling of vitreous obtained from patients who underwent vitrectomy for macular hole surgery.

Results:

apart from known soluble factors in the vitreous

(VEGF, IL6, MCP1, etc), novel factors detected were FasL, Eotaxin, IL23, ITAC, etc. Their presence was correlated with clinical findings and infiltrating immune cell types and their gene expression.

Conclusion:

The data reveal novel angiogenic and inflammatory pathways during disease presentation and therapeutic response.

Introduction

Recent investigations of the tumor microenvironment have shown that many tumors are infiltrated by inflammatory and lymphocytic cells. Increasing evidence suggests that the number, type and location of these tumor-infiltrating lymphocytes in primary tumors has prognostic value, and this has led to the development of an ‘immunoscore. As well as providing useful prognostic information, the immunoscore concept also has the potential to help predict response to treatment, thereby improving decision- making with regard to choice of therapy. This predictive aspect of the tumor microenvironment forms the basis for the concept of immunoprofiling¹ which can be defined as “using an individual’s immune system signature to predict the response to therapy”. The immunoprofile of an individual can be genetically determined or disease induced (and therefore dynamic).

With the above background, lets consider the posterior segment of the eye (vitreous cavity, retina and choroid) as a microenvironment, which under the duress of a chronic metabolically dysregulated inflammatory disease like Diabetes and its ocular complication, diabetic retinopathy may have specific immunoprofile and exploring this may help in understanding the disease better.

Inflammation induce apoptotic switch may be the reason for the pericyte loss and the cause of microvascular occlusion which sets into motion the ischemic process that we see so commonly in non proliferative diabetic retinopathy  and this ischemia may further activate the vascular and inflammatory pathways which may further dysregulate or cause progression of the disease from non proliferative to proliferative retinopathy in a positive feed back loop 

Methods

Cohort details

The study approved by institutional ethics committee and samples from the subject were collected after informed consent. We collected the vitreous samples, of patients who underwent vitrectomy for Diabetic tractional retinal detachment (TRD) and also of patients who underwent Macular hole surgery as which was used as control.  All the samples were cryopreserved until further use. Total of 15 diabetic TRD, and 15 control vitreous sample from macular hole patients were obtained.

Soluble factor profiling and immunophenotyping

The samples were thawed and a comprehensive immunoprofiling (cellular and soluble factors) was performed by isolating the cellular content and acellular content of the vitreous by centrifugation. Secreted / soluble factors (cytokines, chemokines, cell adhesion molecules, growth factors) were measured by multiplex ELISA using flow cytometer based “Cytometric Bead Array”. The concentration of the soluble factors was normalized to the total protein content in the acellular content of the vitreous humor. The total protein content was measured by BCA analysis. Type of immune cells, particulary innate immune cells in vitreous humor was identified, their sub-type frequencies were measured by flow cytometry based immunophenotyping, a procedure that involves the using of fluorochrome conjugated cell type specific antibodies.

Results

A significant increase in the levels of VEGF, PDGF-AA, EPO, FasL and MMP9 was observed in the vitreous of TRD patients compared to the control group. Furthermore, cytokines, chemokines and soluble cell adhesion molecules such as IL-1, IL-4, IL-6, IL-8, IL-13, MCP1 and ICAM1 were significantly higher in the patients compared to control groups.

The vitreous humor exhibited presence cells and it was confirmed that there was significant increase in the level of leukocytes (CD45+ cell) in the vitreous humor of patients compared to controls. Furthermore, significantly higher levels of neutrophils (CD66b+ cells), activated neutrophils (CD66b+ high expression cells), macrophages (CD163+ cells) and natural killer cells (CD56+ cells) were observed in the patient group compared to controls.

This suggests the presence of aberrant inflammatory state along with immune cell influx in the vitreous humor of patients compared to controls.

Discussion

Molecular factor profiling in the aqueous and vitreous humors in retinal conditions like DR have added considerable knowledge in understanding disease pathogenesis, treatment outcomes and the possibilities for novel therapeutics. However, the extent of factors that have profiled had been limited. In this study we have profiled, around 50 such soluble factors along different classes such as cytokines, chemokines, growth factors and cell adhesion molecules. The profiled molecular factors have been shown to have an influence on pathological angiogenesis, either in ocular conditions or diseases of other organ systems. Hence,the current findings will be useful in staging DR, its response to therapy and under standing its pathobiology. Along with well-known dysregulated factors, unique to DR such as VEGF and IL-6, we have observed dysregulation in novel factors such as angiogenin, ICAM1. Also the matrix metallo-proteinases MMP9, MMP2 were found to be significant higher in the PDR group.

Suggesting that there was significant inflammation in the PDR group along with up regulation of vascular growth factors. Cytokines are cell signaling molecules composed of proteins that are secreted by many different cells in the organ tissues. The cytokines can interact with the cells of the immune system and other tissues in the organ to regulate the body’s response to disease, infection or injury. They also regulate inflammatory response which can impact the status of the disease. Chemokines and cell adhesion molecules are a class of soluble factors which was observed to be altered in DR, these factors facilitate the influx or migration of immune cells to the site of injury or interest. Further, they have also been shown to impact pathological angiogenesis various in vitro and animal model systems. The primary knowledge regarding inflammatory microenvironment in the vitreous has primarily been from the knowledge obtained about the levels of various soluble factors concentration.

There has been extremely sparse information related to the immune cell profile in the vitreous that contributes to the inflammatory status in DR. The current study provides very critical information on the frequencies and subtypes of immune cells including neutrophils and macrophages in the vitreous cavity. The role of neutrophils and macrophages have been reported to impact disease pathogenesis and treatment outcome in various in vivo studies. The dysregulation of these additional factors indicates the potential role of these factors listed in the results section to have an effect on disease progression and its response to treatment.Hence, the information from both immune cell type and immune soluble factors can collectively provide much critical information such as “immune score” which on longitudinal follow ups can be used to predict the prognosis and treatment efficacy in the DR. However, a larger cohort and longitudinal follow up with all clinical parameters will provide a wealth of knowledge which will revolutionize our understanding of DR and its management.

Conclusion

Dysregulated inflammatory microenvironment in the vitreous cavity of diabetic retinopathy patients. This aberrant environment can contribute to disease progression and impact response to therapy. The data reveals novel angiogenic and inflammatory pathways as indicated by the factors that were identified which may have a role in Proliferative Diabetic retinopathy during disease presentation and the need for search for newer drugs for a better therapeutic response and optimal management of Proliferative diabetic disease in the future.

References

1. Gass JDM. Stereoscopic Atlas of Macular Diseases. St Louis,MO: Mosby; 1997.
2. Yanuzzi LA. Type-A behavior and central serous retinopathy.Retina 1987;7:111–130.
3. Haimovici R, Gragoudas ES, Duker JS, et al. Central serous chorioretinopathy associated with inhaled or intranasal corticosteroids.Ophthalmology 1997;104:1653–1660.
4. Bouzas EA, Scott MH, Mastorakos G, et al. Central serouschorioretinopathy in endogenous hypercortisolism. Arch Ophthalmol1993;111:1229–1233.
5. Guyer DR, Yannuzzi LA, Slakter JS,et al.Digital indocyanine green videoangiography of central serous chorioretinopathy.Arch Ophthalmol.1994Aug;112(8):1057-62.
6. Iijima H, Iida T, Murayama K, Imai M, Gohdo T. Plasminogen activatorinhibitor 1 in central serous chorioretinopathy. Am J Ophthalmol. 1999127:477–478.
7. Yadav NK, Jayadev C, Mohan A, et al.Subthreshold micropulse yellow laser (577 nm) in chronic central serous chorioretinopathy: safety profile and treatment outcome. Eye (Lond). 2015 Feb;29(2):258-64; quiz 265.
8. Kim JY, Park HS, Kim SY.Short-term efficacy of subthreshold micropulse yellowlaser(577-nm) photocoagulation for chronic central serous chorioretinopathy.Graefes Arch Clin Exp Ophthalmol. 2015 Dec;253(12):2129-35.
9. Ambiya V, Goud A, Mathai A, et al.Microsecond yellow laser for subfoveal leaks in central serous chorioretinopathy.Clin Ophthalmol. 2016 Aug 11;10:1513-9.
10. Maruko I, Koizumi H, Hasegawa T, et al.Subthreshold 577 nm micropulse lasertreatment for central serous chorioretinopathy.

PLoS One. 2017 Aug 29;12(8):e0184112.

11. Arsan A, Kanar HS, Sonmez A. Visual outcomes and anatomic changes after sub-threshold micropulse yellowlaser(577-nm) treatment for chronic central serous chorioretinopathy: long-term follow-up.Eye (Lond). 2018 Apr;32(4):726-733.
12. Ozmert E, Demirel S, Yanık O, et al.Low-Fluence Photodynamic Therapy versus Subthreshold Micropulse YellowWavelength Laserin the Treatment of Chronic Central Serous Chorioretinopathy.J Ophthalmol. 2016;2016:3513794.
13. Roca JA, Wu L, Fromow-Guerra J, Rodríguez FJ, et al.Yellow(577 nm) micropulse laserversus half-dose verteporfin photodynamic therapy in eyes with chronic central serous chorioretinopathy: results of the Pan-American Collaborative Retina Study (PACORES) Group.Br J Ophthalmol. 2018 Feb 8. pii: bjophthalmol-2017-311291.