Cytokines: Made Simple for the Ophthalmologists

Dr. Swathi Lingam, PhD

In the time of the global pandemic known as COVID- 19, everyone has heard the phrase ‘cytokine storm’ and how it can be a deadly consequence of the SARS- CoV2 virus infection. What are these cytokines though? What do they normally do in our bodies and why are they sometimes so deadly?

Little multifunctional molecules

The presence of pus-filled exudates, fever, localized swelling, and pain have been well documented since ancient times. Scientists in the 1940’s asked the question: “What causes all these symptoms?” They studied the pus, found that the white blood cells released ‘soluble factors’1 and the rest, as they say, is history.

Structurally, the ‘soluble factors,’ also known as cytokines, are really small proteins (peptides). They are known as cell signaling molecules, as they essentially act as messengers that tell cells what to do. Their general mode of action is quite similar to a domino effect. Cell 1 is stimulated by some external factor and in response it secretes cytokines. These bind to specific cell surface receptors on cell 2 and activate it, which in turn triggers a biological function for cell 2. It is important to note, however, that depending on the function or requirement, cytokines secreted by cell 1 can act on the same cell (autocrine effect), a nearby cell 2 (paracrine effect) or a faraway cell 2 (endocrine effect).2

Role in human physiology and pathology

Cytokines are broadly classified into six groups: Interleukins, interferons, tumour necrosis factors, colony-stimulating factors, chemokines, and transforming growth factors. This is a generic classification, but in reality, there is a lot of overlap. For instance, interleukin 8 (IL-8) is also considered a pro-inflammatory chemokine and is often designated as CXCL8. Cytokines have complex functional networks due to variability in cytokine secretion rates and the presence of cell membrane receptors. Their function can be pleiotropic – meaning one cytokine can have different effects on different cell types, and they work in synergy to produce a biological effect.3 Their roles in normal human physiology are summarised in Table 1.

CYTOKINES

ACTION

Chemokines (CXC, CC, CX3C, XC)

Direct cell adhesion, migration and activation

Haematopoietins/ Colony stimulating factors (G, GM, M, erythropoietin)

Promotion of cell differentiation and proliferation,

Interferons (a, b, g)

Antivirals, activation of macrophages, enhancement of neutrophil and monocyte function

Interleukins (IL 1-12)

Activation, proliferation and differentiation of B & T cells and natural killer cells, production of IgA, IgE, IgG and IgM.

Transforming growth factor b

Inhibition of B & T cell proliferation and activity, promotion of wound healing

Tumour necrosis factors (a, b)

Tumour cytotoxicity, phagocytic cell activation, cachexia

Table 1: Summary of the physiological functions of cytokines.

A detailed description of cytokine function is beyond the scope of this article, so I will stick to their most popular role: Regulating the immune and inflammatory response. To elucidate this in a simplified manner, imagine that a hypothetical patient X has been infected with SARS-CoV2. The virus injects its RNA genome into host lung cells using the ACE2 receptor as an entry point. There, it hijacks the human protein production machinery and rapidly multiplies, producing many daughter viruses.

Typically, at this point, the innate immune response would kick in. Infected cells produce cytokines called interferons, which, as the name suggests, interfere with the viral proliferation and also signal to neighboring cells that there is an infection. Cytotoxic T cells and natural killer (NK) cells are also recruited to the infection site through other mechanisms and kill the infected cells. The cytotoxic cells also release more cytokines to enhance the immune response by stimulating the infected cell or its neighbours to recruit more NK cells/ cytotoxic T cells.4

What happens if the innate immune response does not do its job? There is evidence that SARS-CoV2 is able to evade the innate immune response and proliferate undetected by the immune system.5 It is now up to the adaptive immune response to fight off the virus. It does this on two fronts. One, the infected cell can present viral antigens on its membrane, which allows antibodies to attack the cell directly, supported by specialist white blood cells (B cells). Alternatively, the daughter viruses that burst out of infected cells are targeted by antibodies that trap the virus in complexes that are ‘eaten’ or phagocytosed by specialized cells called macrophages. 4

The recruitment and activity of these various cells is tightly controlled by cytokines. For instance, interleukins are essential for the activation and proliferation of the cells involved in both immune responses. They also stimulate B cells to produce antibodies.6 The action of killing cells and eating antibody- virus complexes also causes inflammation, which is essential for the support and augmentation of the immune response. Inflammation normally manifests as fever and can be harmful if not tightly controlled. Like everything else, this response is mediated by cytokines. Proinflammatory cytokines stimulate inflammation and in order to maintain homeostasis, anti-inflammatory cytokines inhibit the signaling and limit inflammation.4 When the immune system is working in overdrive to eliminate the virus, there can sometimes be a loss of this delicate balance. The inflammation response becomes uncontrolled and there is a sudden burst of cytokines. They start attacking other organs in the body, resulting in multiorgan failure and eventually death. This uncontrolled cytokine release is more popularly known as a ‘cytokine storm’. This phenomenon is unfortunately poorly understood at this point.7

Aberrant cytokine activity is also associated with chronic inflammatory and autoimmune pathologies such as Alzheimer’s, type 1 diabetes, and Rheumatoid arthritis. Dysregulation of interleukins and tumor necrosis factor (TNF) are most commonly associated with these diseases.6 To understand this dysregulation further, let us consider the example of type 1 diabetes (T1D). This is an autoimmune disease characterized by pancreatic b cell loss and hyperglycemia. One of the drivers of b cell loss is inflammation, and this is mediated by cytokines. Just as in the inflammatory response, some cytokines protect b cells and maintain immune tolerance, while others promote the differentiation and pancreatic infiltration of damaging immune cells; causing the onset of T1D.8

Cytokines in ophthalmic pathologies

Ocular cells that can produce cytokines include the retinal pigment epithelium (RPE), Müller cells, lens epithelial cells, corneal stromal and epithelial cells, and ciliary body epithelial cells. The RPE for instance is at the crux of retinal immunity and its dysregulation in age-related macular degeneration (AMD) is known to result in localized, cytokine-mediated chronic inflammation that usually exacerbates the disease.9 In another interesting example of inflammation playing a role in the development of ocular disease, inflammatory cytokines including IL-6 were associated with the development and progression of high myopia and myopic retinopathy.10

Similar to the T1D example above, uveitis is an autoimmune disease that is influenced by cytokines produced by T helper cells. For instance, IL-6, IL-17, and TNFa are known to exacerbate the symptoms, whereas IL-23 is thought to have a protective effect.11 Cytokines produced by T helper cells are also involved in another common inflammatory disease: Dry eye. Through a tug of war, IL-13 that regulates goblet cell differentiation is antagonized by interferon y, which promotes apoptosis of epithelia on the ocular surface. This is made worse by the action of Il-17, which promotes disruption of the corneal epithelial barrier.12

Cytokine modulation in disease treatment

Due to their role in the inception and development of various debilitating and fatal conditions, it important to develop therapies that can control cytokine levels in the body. The most obvious way to accomplish this is to design agents that will specifically block cytokine activity. Hence, the typical strategy is to either block the cytokine or its receptor. Blocking the cytokine is typically accomplished by using neutralizing antibodies, or soluble cytokine scavengers that mimic cytokine receptors. Anti- receptor strategies include using receptor antagonists (drugs that preferentially bind to cytokine receptors, hence preventing cytokine binding) and inhibitors of cytokine- regulated intracellular pathways. Anti- cytokine therapies are currently being used in the clinic to treat several autoimmune diseases, cancers, asthma and for bone marrow suppression.13

Conclusion

Cytokines are small molecules, but they are mighty. They are essential for maintaining the delicate balance of the human immune system amongst other functions. They can even influence vaccine efficacy and allograft acceptance. Disruption of their regulatory function leads to lifelong chronic, debilitating diseases. With a deeper understanding of their structure and function, it is now possible to develop therapies that can modulate/ inhibit their effect on these diseases. Although the results have been a mixed bag, the success stories have been promising. With more research and clinical trials, anti-cytokine therapies could be the best way forward to combat these intractable conditions.

Bibliography

1.Dinarello, C. A. Historical Review of Cytokines. Eur. J. Immunol. 37, S34–S45 (2007).
2. Altan-Bonnet, G. & Mukherjee, R. Cytokine-mediated communication: a quantitative appraisal of immune complexity. Nat. Rev. Immunol. 19, 205–217 (2019).
3. Testar, J. Cytokines : Introduction. Available at: http://bitesized.immunology.org/receptors-and-molecules/cytokines/.
4.Mueller, S. N. & Rouse, B. T. Immune responses to viruses. Clin. Immunol. 421–431 (2008). doi:10.1016/B978-0-323-04404-2.10027-2
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6. Turner, M. D., Nedjai, B., Hurst, T. & Pennington, D. J. Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease. Biochim. Biophys. Acta - Mol. Cell Res. 1843, 2563–2582 (2014).
7. Shimabukuro-Vornhagen, A. et al. Cytokine release syndrome. Journal for ImmunoTherapy of Cancer 6, (2018).
8.Lu, J., Liu, J., Li, L., Lan, Y. & Liang, Y. Cytokines in type 1 diabetes: mechanisms of action and immunotherapeutic targets. Clin. Transl. Immunol. 9, 1–17 (2020).
9.Wooff, Y., Man, S. M., Aggio-Bruce, R., Natoli, R. & Fernando, N. IL-1 family members mediate cell death, inflammation and angiogenesis in retinal degenerative diseases. Front. Immunol. 10, 1–21 (2019).
10. Yuan, J. et al. Inflammatory cytokines in highly myopic eyes. Sci. Rep. 9, 1–9 (2019).
11. Weinstein, J. E. & Pepple, K. L. Cytokines in uveitis. Current Opinion in Ophthalmology 29, 267–274 (2018).
12. Pflugfelder, S. C., Corrales, R. M. & de Paiva, C. S. T helper cytokines in dry eye disease. Experimental Eye Research 117, 118–125 (2013).
13.Ramani, T. et al. Cytokines: The Good, the Bad, and the Deadly. International Journal of Toxicology 34, 355–365 (2015).

The author is a regular blogger. You can read her blog The Very Curious Biochemist and follow her on Twitter.

Dr. Swathi Lingam, PhD
Research fellow, A*Star, Singapore
Swathi Lingam is a research fellow at A*Star in Singapore, where she is developing novel cell therapy systems to treat age-related eye diseases. She had been a Ph.D. student at the University of Manchester, followed by a short post-doc at the University of Oxford before moving to Singapore. She is passionate about science communication and loves doing that through her blog The Very Curious Biochemist. Follow her on Twitter.
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