*4.4.3.2 IL-1*β

Macrophages produce IL-1β which is an inflammatory cytokine mainly which can further activate the transcriptional factor NF-κB, which plays an important role in transcription of inflammatory cytokines [92]. Furthermore TNF-α and recombinant IL-1β seem to stimulate human retinal pigment epithelium cells leading to secretion of IL-6 and IL-8 [93]. It has been seen in studies that TNF-α and IL-1β promote angiogenic activity leading to stimulation and synthesis of collagen glial cells, and fibroblasts leading to proliferation and contraction promoting angiogenesis and ocular neovascularization [94]. It has been observed that invading microorganisms activate the inflammatory response by secreting pro-inflammatory cytokines particularly IL-1β. IL-1-responsive genes initiate and coordinate local inflammation and also attract and activate cells of the adaptive immune system at sites of infection eventually leading these signals to activate NALP3-inflammasome pathway, which plays a central role in acute and chronic sterile inflammation [95]. Studies assessing inflammatory mechanisms involving NLRP3 inflammasome were carried out using akimbo mouse, revealing an increased vascular leakage, reduced retinal thickness, and function in Akimba retina. High levels of IL-1β along with increased NLRP3, ASC, and Caspase-1 at mRNA and protein levels were seen suggesting a critical role for NLRP3 inflammasome in akimbo retina depicting advanced stages of DR pathogenesis [96]. Other studies have shown elevated levels in aqueous of IL-6 and macular thickness indicating IL-6 may play a central role in the development of diabetic macular edema [97].

## *4.4.3.3 TNF-alpha*

TNF-α, a cytokine with tumour necrosis activity is produced by various types of cells which includes macrophages, is recognised as an important host defence factor that affects malignant and normal cells. It is synthesised by T cells and macrophages and its expression is regulated by NF-κB [98]. It also plays the role of inflammatory mediator of neuronal cell after cerebral ischemic trauma and also a similar role in retinal tissue [99]. Besides increasing endothelial cell permeability [100]. TNF-α is also involved in stimulating leukocyte adhesion and inducing oxidation and simultaneous production of reactive oxygen due to the death of retinal ganglion cells and degeneration of the optic nerve [101]. High pharmacological doses of TNF-α combined with chemotherapy has been seen to regress intractable tumours. Evidence demonstrates that pathophysiological concentrations of endogenous TNF-α could act to promote tumour genesis and growth [102]. In diabetic retinopathy pro-inflammatory mediators regulated by cytokines, such as TNF-α, IL-1β and growth factors leads to further progression of these processes, leading to vasopermeability (diabetes macular edema) and/or pathological angiogenesis (proliferative diabetic retinopathy) [103]. Diabetic patients have shown higher TNF-α levels in vitreous/serum ratio compared to non-diabetics [104]. Strong correlation between plasma TNF-α levels and severity of DR has been documented [105]. It has been documented that TNF-α is expressed in the endothelial cells and stromal cells of the fibrovascular membranes of diabetic patients with PDR [106]. Studies have confirmed the presence of vascular endothelial growth factor (VEGF) and TNF-α in epiretinal membranes in proliferative eye disease [107]. Recent studies assessing the impact of anti-TNF agents on intermediary metabolism suggest that TNF-α blockade could improve insulin resistance and lipid profiles in patients with chronic inflammatory disease [108].

#### *4.4.3.4 HMGB1*

HMGB1 though secreted from numerous sites in the retina, including the ganglion cell layer, inner nuclear layer, outer nuclear layer, inner and outer segment of the photoreceptors, and retinal pigment epithelial cells [109, 110]. It is a protein that stabilises the formation of nucleosomes and gene transcription [111]. Studies indicate that HMGB1 is released from activated innate immune cells or necrotic cells and functions as an important mediator of endotoxaemia, sepsis, arthritis, and local inflammation hence agents that inhibit HMGB1 release or action, confer significant protection against endotoxaemia, sepsis, and arthritis in animal models and thus hold potential for the clinical management of various inflammatory diseases [112]. HMGB1 functions as a cytokine that amplifies the effect of the receptor for AGE

(RAGE) axis and mediates the secretion of survival factors such as VEGF-A, to counteract the effects of oxidative stress. HMGB1 is thought to contribute to the accelerated micro and macro-vasculopathy seen in diabetes [113]. Its level has been detected on higher side in vitreous in patients with PDR and has been detected in endothelial and stromal cells of ERM in PDR patients [114].
