Review Ageing and ocular surface immunity Alireza Mashaghi, Jiaxu Hong, Sunil K Chauhan, Reza Dana

Schepens Eye Research ABSTRACT infection and autoimmunity as well as to increased Institute, Massachusetts Eye The prevalence of ocular surface immunopathologies is severity of autoimmunity.10 Age-related immune and Ear Infirmary, Harvard enhanced in the elderly. This increased prevalence has been system changes affect both the innate11 and the Medical School, Boston, 12 Massachusetts, USA attributed to age-related dysregulation of innate and adaptive arms of immunity. A decrease in nearly adaptive immune system responses. Age-related changes in all innate Toll-like receptor (TLR)-induced Correspondence to ocular surface immunity have similar and distinct responses and higher levels of many proinflamma- Dr Reza Dana, Schepens Eye characteristics to those changes seen in other mucosal tory cytokines are observed in the elderly.10 The Research Institute, fi Massachusetts Eye and Ear tissues. This mini review provides a brief outline of key bene ts of vaccination to prevent infectious disease Infirmary, Harvard Medical findings in the field of ocular ageing, draws comparisons are also limited in the elderly, predominantly due School, 20 Staniford Street, with other mucosal tissues and, finally, discusses age-related to the inability to maintain long-term adaptive Boston, MA 02114, USA; changes in the context of immunopathogenesis of infectious immune responses.13 Age-related deficiencies in [email protected] keratitis and dry eye disease, two of the most common maintaining telomeres and DNA stability cause Received 14 September 2015 inflammatory disorders of the ocular surface. excessive apoptosis of lymphocytes. This process Revised 26 April 2016 can add to the severity of certain diseases. For Accepted 19 June 2016 example, in rheumatoid arthritis, increased apop- Published Online First tosis of naive T cells leads to impairment of T cell 4 July 2016 INTRODUCTION With time, the human body loses many homeostatic regeneration and a dramatic change in the T cell mechanisms, leading to increased vulnerability to repertoire; suppressing this phenomenon by reju- organ dysfunction and ultimately death. Ageing not venating the immune system reduces the severity of only leads to body dysfunction per se, but also the disease.9 Table 1 summarises age-related enhances the susceptibility to foreign invaders such changes in the frequencies and functions of as viruses and bacteria due to dysfunction or dysre- immune cells. gulation of the immune system.1 What molecular Alterations of mucosal immunity by age have and cellular mechanisms underlie ageing? been documented both in human and animal Hallmarks of ageing include genomic instability, models. A reduction in lymphoreticular tissues, telomere attrition, epigenetic alterations, loss of pro- antigen-specific IgA antibody responses and lack of teostasis, dysregulated nutrient sensing, mitochon- oral tolerance induction are three hallmarks of drial dysfunction, cellular senescence, stem cell mucosal ageing in the gastrointestinal track.14 15 exhaustion and altered intercellular communica- Peyer’s patches in the gastrointestinal mucosa tion.2 For postmitotic cells, such as neurons and shrink with age and have reduced frequencies muscle cells, these processes lead to a gradual loss of of naive CD4+ T cells and dendritic cells (DCs).16 normal structure and function, the so-called In the respiratory system, though ‘chronological ageing’.3 For continuously dividing nasopharyngeal-associated lymphoreticular tissue cells, like those of the epithelia of the skin or gut, remains intact during ageing,14 age-related immune ‘replicative ageing’ further challenges the function deficits, known as immunosenescence, have been of tissues in which these cells reside. Replicative shown to increase respiratory infections.17 A diffe- ageing refers to the accumulation of cellular rent onset of immunosenescence in gastrointestinal, damage, such as telomere shortening and nasopharyngeal and ocular mucosa has been replication-associated DNA mutations, that occurs reported, but the general immune response is during the process of cell division.34Despite similar similar in young and old.14 trends,5 interindividual variations in ageing are sig- For the ocular mucosa, a plausible theory based nificant.67Precisely what causes the heterogeneity on the above observation in other tissues is that the in the rate of progression and the onset of rapidity and specificity of the immune response in age-related dysfunctions remains largely unknown. both the inflammatory and regulatory arms of the Understanding the ageing process, though limited, immune system are reduced with ageing. As we allows for novel diagnostic and therapeutic interven- discuss in the subsequent sections, this reduction tions. In a number of model organisms, the ageing may lead to autoimmune disease and increased process was observed to slow down or even arrest tissue damage as a result of infection. We also temporarily. A number of genes as well as environ- discuss how age-related changes in the frequencies mental factors (eg, dietary restriction) appear to of ocular immune cells and their expression of extend not only the life span, but also maintain various cytokines and chemokines18 are associated health.8 Rejuvenating the immune system has turned with increased autoimmunity. out to be a real possibility, with beneficial impact on 189 the progression and severity of diseases. AGEING AND OCULAR SURFACE IMMUNITY fi To cite: Mashaghi A, The tear lm, lacrimal glands, corneal and conjunc- Hong J, Chauhan SK, et al. AGEING AND THE IMMUNE SYSTEM tival epithelia, and meibomian glands ensure the Br J Ophthalmol The immune system undergoes profound changes integrity and function of the ocular surface.19 Studies – 2017;101:1 5. with age, which leads to higher susceptibility to using human subjects as well as animal models

Mashaghi A, et al. Br J Ophthalmol 2017;101:1–5. doi:10.1136/bjophthalmol-2015-307848 1 Review

response that involves T cells. The destructive nature of HSV Table 1 Age-associated changes in the immune system infection has been attributed primarily to the CD4+ Th1-type Innate inflammatory immune response within the cornea rather than a Neutrophils Reduced function including chemotaxis, microbial killing cytopathic response elicited by the virus itself.43 For a given and phagocytosis. load of virus at the eye or trigeminal ganglion, old mice show a Macrophages Defective chemotaxis, cytokine production and more severe keratitis.43 phagocytosis HIV infection is another viral infection that affects the ocular DCs Change in the balance of plasmacytoid DCs and myeloid surface. HIV-infected individuals undergo accelerated biological DCs 45 Natural killer cells Decreased proliferationReduced production of TNF-α, ageing mediated by increased cellular senescence. For IL-2, IL-12 and IL-2R example, corneal endothelial cells in patients with HIV show Adaptive increased variation in cell size and lower cell density, consistent B cells Reduced B cell lymphopoiesis; reduced CD4+ T cell help; with HIV-related accelerated senescence, especially among those reduced quantity and quality of antibodies with poor immune recovery.46 The effect of HIV on other cell CD4+ T cells Reduced IL-2 production; dampened co-stimulation types and resident immune cells is poorly understood and CD8+ T cells Reduced repertoire; increased frequencies of memory remains to be investigated; however, it is known that both HIV cells infection and ageing are characterised by a deficiency in the Tregs Increased frequencies number of competent T cells. Recent studies show that antiretro- For more information, please see McKay et al89 and references therein. viral therapy, despite the adequate recovery of CD4+ T cell DCs, dendritic cells; IL, interleukin; TNF, tumour necrosis factor. counts and suppression of viremia, is less efficient in restoring the immune system in older patients.47 HIV-infected children exhibit premature biological ageing with accelerated immune suggest that these guardians of the ocular surface integrity senescence, which particularly affects the CD8+ cell subset. are affected in the course of ageing; for example, the lacrimal HIV infection itself also seems to influence the ageing process – gland is affected due to exposure to oxidative stress.20 27 rather than exposure to antiretroviral therapy for prophylaxis or Age-related physiological changes of the ocular surface have been treatment.48 The influence of HIV on accelerated senescence of – reported;25 28 however, the effect of ageing on ocular surface ocular cells can be complicated by a number of associated path- immunity remains poorly understood. Immunological mechanisms ologies, such as Kaposi sarcoma, a highly vascularised tumour, play a pivotal role in regulating the ocular surface environment as as well as with HSV keratitis, fungal keratitis (eg, Candida para- well. Immune cells directly or via secretion of immunomodulatory psilosis and Candida albicans) and uveitis, which are more factors actively protect the ocular surface.19 Resident corneal severe in elderly HIV-infected patients.49 50 antigen-presenting cells,29 30 regulatory T cells31 32 and T helper 1 Bacterial keratitis is a serious corneal infectious disease that can (Th1) cells33 are among key cellular players in immune homeosta- result in severe visual disability. Pseudomonas aeruginosa infection sis. Regulatory T cells, for example, suppress autoreactive effector in aged mice induces increased tissue damage compared with T cells protecting against excessive adaptive immunity. young mice.51 This difference has been attributed to the persist- Furthermore, immunomodulatory factors such as vascular endo- ence of polymorphonuclear neutrophils (PMNs) in the cornea of thelial growth factor receptor-3, transforming growth factor-β34 35 old mice and contributes to increased corneal tissue destruction. programmed death-ligand 1, interleukin-1 receptor antagonist An increased production of chemoattractant macrophage inflam- and interleukin (IL)-1336 regulatetheimmunemicroenvironment matory protein 2 (MIP-2) likely underlies the altered PMN of the healthy ocular surface and inhibit immunopathogenic response in these older animals.51 Neutrophils generate a number mechanisms.37 38 A breakdown of this immunological balance of proteases and oxidases that work together to digest the ocular impairs the function of the eye and the visual system. surface and in particular the cornea.52 Overall, alterations of the Immunosenescence is of great importance in a number of innate system associated with ageing may increase morbidity ocular surface pathological conditions including infection, auto- caused by bacterial infection, yet the role of ageing in the immuno- immunity and ocular complications of systemic autoimmunity senescence of adaptive immunity remains elusive. such as graft-versus-host disease (GVHD), systemic lupus erythe- matosus and Sjögren’s disease. It is known that the incidence of Ageing and dry eye disease viral, bacterial and fungal keratitis and conjunctivitis is also Ocular surface autoimmune diseases comprise a diverse spec- increased in the elderly.39 Additionally, several studies have iden- trum of pathologies and can be classified as ocular specific (eg, tified age as one of the risk factors for GVHD40 and Sjögren’s dry eye, Mooren’s ulcerative keratitis) or systemic (eg, Sjögren’s disease.41 In this section, we provide an overview of how the syndrome, cicatricial pemphigoid, rheumatoid arthritis, systemic ocular surface immunity changes with ageing and how these lupus erythematosus).53 A common immune-mediated disorder affect infectious and autoimmune disorders.31 of the ocular surface is dry eye disease (DED). DED is known to disrupt the integrity of the corneal epithelium, leading to expos- ure or altered expression of self-antigens, breakdown of corneal Ageing and infectious keratitis immune privilege and a T cell-mediated autoimmune response – Infectious keratitis in the elderly tends to be more severe with a directed against the ocular surface.19 32 54 61 Based on our worse clinical outcome compared with young patients.42 Ocular current understanding, the principal inducers in the early surface infection ignites different immune responses that act to immune response of DED are antigen-presenting cells, primarily control the infection, but may also lead to tissue destruction. resident ocular surface DCs.19 30 They present ocular surface Herpes simplex virus (HSV) infection, the leading infectious antigens to T cells in the draining lymph nodes, where naive cause of blindness in the USA, shows an increased incidence in T cells are primed and expanded as CD4+ Th 1 and Th17 – the elderly.43 HSV infection typically activates an early innate effector cells.62 64 Both CD4 T cell subsets have been shown to immune response (increased expression of TLR7 and neutrophil contribute to the development of ocular surface inflammation in – chemokine IL-8 by epithelial cells44), followed by an adaptive DED.59 65 74 The observations of (i) increased Th17 cells in the

2 Mashaghi A, et al. Br J Ophthalmol 2017;101:1–5. doi:10.1136/bjophthalmol-2015-307848 Review lymph nodes of DED mice and that (ii) in vivo neutralising of antigen-presenting cell migration and the Treg/Th17 balance can IL-17 restores Treg function and inhibits the induction and pro- be extended to the ocular surface and DED or not remains to be gression of DED implicate Th17 cells as a critical CD4 effector explored. We hypothesise that a higher inflammatory reaction of cell population mediating DED.31 59 Participation of Th1 in the DED in the elderly could be explained by the enhancement – pathogenesis of DED has been demonstrated as well.59 65 67 75 of antigen-presenting process, accumulated memory T cells Increased interferon (IFN)-γ has been found in the conjunctiva and an imbalance in the number and/or function of Treg and of patients with DED and in biopsies taken from patients with Th17 cells with a high basal level of proinflammatory mediators Sjögren’s syndrome. In animal models, IFN-γ has been impli- among the aged. cated in conjunctival and corneal epithelial apoptosis and loss of goblet cells, and neutralisation of IFN-γ has blocked dry eye CONCLUDING REMARKS induction. Despite involvement of IFN-γ, it is yet to be proven In this review, we discussed evidence that indicates the critical whether Th1 cells are the primary source of IFN-γ in DED effects of ageing on the regulation of innate and adaptive since macrophages and other cells can also express IFN-γ. Other immunity at the ocular surface. Briefly, increased expression of immune cell types have also been implicated in the pathogenesis neutrophil chemokines (eg, IL-8) and Toll-like receptors (eg, 76 of DED including PMNs that regulate Tregs and Th17 cells. TLR7) by epithelial cells may contribute to an enhanced innate Clinical observations suggest that DED has a higher incidence in immune response in the aged ocular surface. In infection, the 77 the elderly. This can be attributed to age-associated decrease enhanced persistence of PMNs and their products, such as pro- in aqueous tear production and age-related alteration of ocular teases and oxidases, may damage the ocular surface in the 78 surface immunity. Although age is associated with increased elderly. Aged DCs are functionally impaired in activation, migra- inflammation and autoimmunity, very few articles are available tion and production of cytokines upon stimulation with foreign on the effects of age on the function of T cell immunity in antigens. However, their reactivity to self-antigens seems to DED. A recent study in mice investigated age-related changes of increase with ageing. In regard to adaptive immune cells, the 18 the ocular immune system. McClellan et al showed increased frequencies and activity of Tregs change in the steady state in CD4 T cell infiltration and enhanced expression of IFN-γ and aged subjects; however, little is known about the dynamics of IL-17A in the conjunctiva. Their data suggest that CD4 T cells Tregs in ocular pathological conditions in these subjects. Th17 from aged mice are more pathogenic and capable of inducing cells and in particular memory Th17 are dramatically elevated, a DED. However, the exact molecular mechanisms underlying the phenomenon that coincides with increased chronicity of ocular effects of ageing on DED remain largely unexplored. surface inflammation in the elderly. Future studies are required Studies on tissues other than the ocular surface may provide to study the altered balance between Treg, Th1 and Th17 cells insights into how ageing affects DED. Researchers have previ- in the elderly and how they enhance the inflammation at the ously analysed the effects of ageing on DCs, Th17 and Treg cells. ocular surface. Overall, it seems that in the aged ocular surface These three cell types are the principal components in DED response to infections is impaired, resulting in immunodefi- pathogenesis. It is known that in the course of ageing DCs are ciency, whereas increased reactivity to self-antigens results in functionally impaired in many aspects, including activation, chronic inflammation and autoimmunity. migration and production of cytokines in response to stimuli.79 In contrast to their reduced functions with ageing, an increased Acknowledgements The authors thank Dr Susanne Eiglmeier for critical reading reactivity of aged DCs against self-antigens has been observed.80 the manuscript. In addition, DCs from the elderly show enhanced inflammatory Contributors AM and JH contributed equally to the article. AM, JH, and RD cytokine production81 and nuclear factor-κB activation, suggest- designed and drafted the paper. AM, JH, SKC and RD reviewed and revised the paper. ing that these DCs are in a relatively activated state.82 In addition, frequencies and function of T cells are affected by age. The Funding Supported in part by National Institute of Health grants EY020889 (RD) and EY024602 (SKC). numbers of murine Tregs increase with age in the spleen and – lymph nodes.83 85 In humans, skin Treg numbers are increased in Competing interests None declared. the steady state of aged subjects, and functional changes in Treg Provenance and peer review Not commissioned; externally peer reviewed. may occur with ageing.86 We note that an increase in the number of Tregs does not necessarily imply an increase in Treg function. REFERENCES Indeed, autoimmunity is more common in the elderly and that 1 Dorshkind K, Montecino-Rodriguez E, Signer RAJ. 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