Aged Mice Exhibit Severe Exacerbations of Dry Eye Disease with an Amplified Memory Th17 Cell Response
2020; Elsevier BV; Volume: 190; Issue: 7 Linguagem: Inglês
10.1016/j.ajpath.2020.03.016
ISSN1525-2191
AutoresWilliam Foulsham, Sharad K. Mittal, Y. Taketani, Yihe Chen, Takeshi Nakao, Sunil K. Chauhan, Reza Dana,
Tópico(s)Olfactory and Sensory Function Studies
ResumoThe prevalence as well as the severity of dry eye disease increase with age. Memory T helper 17 (Th17) cells (CD4+IL-17A+CD44+) drive the chronic and relapsing course of dry eye disease. Here, we investigated the contribution of memory Th17 cells to age-related dry eye disease, and evaluated memory Th17 cell depletion with anti–IL-15 antibody as a strategy to abrogate the severe exacerbations of dry eye disease observed in aged mice. After initial exposure to desiccating stress, aged mice maintained higher frequencies of memory Th17 cells in the draining lymph nodes relative to young mice. Upon secondary exposure to desiccating stress, aged mice developed more severe corneal epitheliopathy than young mice, which is associated with increased local frequencies of Th17 cells (CD4+IL-17A+). Treatment with anti–IL-15 antibody decreased the enlarged memory Th17 pool in aged mice to frequencies comparable with young mice. Furthermore, anti–IL-15–treated mice showed significantly reduced conjunctival infiltration of Th17 cells and lower corneal fluorescein staining scores compared with saline-treated control mice. Our data suggest that age-related increases in the memory Th17 compartment predispose aged mice toward the development of severe corneal epithelial disease after exposure to a dry environment. Selectively targeting memory Th17 cells may be a viable therapeutic approach in the treatment of age-related dry eye disease. The prevalence as well as the severity of dry eye disease increase with age. Memory T helper 17 (Th17) cells (CD4+IL-17A+CD44+) drive the chronic and relapsing course of dry eye disease. Here, we investigated the contribution of memory Th17 cells to age-related dry eye disease, and evaluated memory Th17 cell depletion with anti–IL-15 antibody as a strategy to abrogate the severe exacerbations of dry eye disease observed in aged mice. After initial exposure to desiccating stress, aged mice maintained higher frequencies of memory Th17 cells in the draining lymph nodes relative to young mice. Upon secondary exposure to desiccating stress, aged mice developed more severe corneal epitheliopathy than young mice, which is associated with increased local frequencies of Th17 cells (CD4+IL-17A+). Treatment with anti–IL-15 antibody decreased the enlarged memory Th17 pool in aged mice to frequencies comparable with young mice. Furthermore, anti–IL-15–treated mice showed significantly reduced conjunctival infiltration of Th17 cells and lower corneal fluorescein staining scores compared with saline-treated control mice. Our data suggest that age-related increases in the memory Th17 compartment predispose aged mice toward the development of severe corneal epithelial disease after exposure to a dry environment. Selectively targeting memory Th17 cells may be a viable therapeutic approach in the treatment of age-related dry eye disease. Aging results in a general decrease in immune function, observed clinically as increased vulnerability to infection1Thompson W.W. Shay D.K. Weintraub E. Brammer L. Cox N. Anderson L.J. Fukuda K. Mortality associated with influenza and respiratory syncytial virus in the United States.JAMA. 2003; 289: 179-186Crossref PubMed Scopus (3027) Google Scholar and impaired responses to vaccination.2Guichelaar T. Hoeboer J. Widjojoatmodjo M.N. Reemers S.S.N. van Els C.A.C.M. Otten R. van Remmerden Y. Boes J. Luytjes W. Impaired immune response to vaccination against infection with human respiratory syncytial virus at advanced age.J Virol. 2014; 88: 9744-9750Crossref PubMed Scopus (15) Google Scholar However, paradoxically, advanced age is also a risk factor for autoimmunity.3Goronzy J.J. Weyand C.M. Immune aging and autoimmunity.Cell Mol Life Sci. 2012; 69: 1615-1623Crossref PubMed Scopus (152) Google Scholar A hallmark of adaptive immunity, immunologic memory permits antigen-experienced lymphocytes to respond rapidly and robustly to repeat exposures to the same antigen.4Weng N.-P. Aging of the immune system: how much can the adaptive immune system adapt?.Immunity. 2006; 24: 495-499Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar Through cognition of self-antigen, immunologic memory also contributes toward chronic inflammation in autoimmunity.5Devarajan P. Chen Z. Autoimmune effector memory T cells: the bad and the good.Immunol Res. 2013; 57: 12-22Crossref PubMed Scopus (65) Google Scholar, 6McGeachy M.J. Th17 memory cells: live long and proliferate.J Leukoc Biol. 2013; 94: 921-926Crossref PubMed Scopus (29) Google Scholar, 7Burkett P.R. Meyer zu Horste G. Kuchroo V.K. Pouring fuel on the fire: Th17 cells, the environment, and autoimmunity.J Clin Invest. 2015; 125: 2211-2219Crossref PubMed Scopus (177) Google Scholar, 8Kryczek I. Zhao E. Liu Y. Wang Y. Vatan L. Szeliga W. Moyer J. Klimczak A. Lange A. Zou W. Human TH17 cells are long-lived effector memory cells.Sci Transl Med. 2011; 3: 104ra100Crossref PubMed Scopus (201) Google Scholar, 9Haines C.J. Chen Y. Blumenschein W.M. Jain R. Chang C. Joyce-Shaikh B. Porth K. Boniface K. Mattson J. Basham B. Anderton S.M. McClanahan T.K. Sadekova S. Cua D.J. McGeachy M.J. Autoimmune memory T helper 17 cell function and expansion are dependent on interleukin-23.Cell Rep. 2013; 3: 1378-1388Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Identifying the role of memory lymphocytes in age-related autoimmune diseases represents a key step in understanding the immunopathogenesis of these chronic inflammatory disorders. Dry eye disease (DED) is an extremely common chronic condition that significantly impairs patient quality of life.10Miljanović B. Dana R. Sullivan D.A. Schaumberg D.A. Impact of dry eye syndrome on vision-related quality of life.Am J Ophthalmol. 2007; 143: 409-415Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 11Crnej A. Kheirkhah A. Ren A. Mullins A. Lavric A. Suri K. Hamrah P. Dana R. Patients' perspectives on their dry eye disease.Ocul Surf. 2016; 14: 440-446Crossref PubMed Scopus (9) Google Scholar, 12Bradley J.L. Özer Stillman I. Pivneva I. Guerin A. Evans A.M. Dana R. Dry eye disease ranking among common reasons for seeking eye care in a large US claims database.Clin Ophthalmol. 2019; 13: 225-232Crossref PubMed Scopus (26) Google Scholar DED is a multifactorial disease of the ocular surface characterized by ocular surface inflammation, loss of tear film homeostasis, and neurosensory dysfunction.13Craig J.P. Nelson J.D. Azar D.T. Belmonte C. Bron A.J. Chauhan S.K. de Paiva C.S. Gomes J.A.P. Hammitt K.M. Jones L. Nichols J.J. Nichols K.K. Novack G.D. Stapleton F.J. Willcox M.D.P. Wolffsohn J.S. Sullivan D.A. TFOS DEWS II report executive summary.Ocul Surf. 2017; 15: 802-812Crossref PubMed Scopus (359) Google Scholar The prevalence of DED increases with age, with a recently published retrospective study of a large US population estimating a prevalence of 0.2% for patients age 2 to 17 years, increasing to 11.7% for patients older than age 50.14Dana R. Bradley J.L. Guerin A. Pivneva I. Stillman I.O. Evans A.M. Schaumberg D.A. Estimated prevalence and incidence of dry eye disease based on coding analysis of a large, all-age United States health care system.Am J Ophthalmol. 2019; 202: 47-54Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar The disruption of ocular surface homeostasis is recognized as the trigger of DED immunopathogenesis.15Guzmán M. Keitelman I. Sabbione F. Trevani A.S. Giordano M.N. Galletti J.G. Desiccating stress-induced disruption of ocular surface immune tolerance drives dry eye disease.Clin Exp Immunol. 2016; 184: 248-256Crossref PubMed Scopus (46) Google Scholar,16Guzmán M. Keitelman I. Sabbione F. Trevani A.S. Giordano M.N. Galletti J.G. Mucosal tolerance disruption favors disease progression in an extraorbital lacrimal gland excision model of murine dry eye.Exp Eye Res. 2016; 151: 19-22Crossref PubMed Scopus (20) Google Scholar A proinflammatory milieu at the ocular surface induces the maturation of antigen-presenting cells, which migrate via afferent lymphatics to the draining lymph nodes.17Stevenson W. Chauhan S.K. Dana R. Dry eye disease.Arch Ophthalmol. 2012; 130: 90Crossref PubMed Scopus (407) Google Scholar, 18Barabino S. Chen Y. Chauhan S. Dana R. Ocular surface immunity: homeostatic mechanisms and their disruption in dry eye disease.Prog Retin Eye Res. 2012; 31: 271-285Crossref PubMed Scopus (212) Google Scholar, 19Foulsham W. Coco G. Amouzegar A. Chauhan S.K. Dana R. When clarity is crucial: regulating ocular surface immunity.Trends Immunol. 2018; 39: 288-301Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 20Schaumburg C.S. Siemasko K.F. De Paiva C.S. Wheeler L.A. Niederkorn J.Y. Pflugfelder S.C. Stern M.E. Ocular surface APCs are necessary for autoreactive T cell-mediated experimental autoimmune lacrimal keratoconjunctivitis.J Immunol. 2011; 187: 3653-3662Crossref PubMed Scopus (118) Google Scholar, 21Pflugfelder S.C. Stern M. Zhang S. Shojaei A. LFA-1/ICAM-1 interaction as a therapeutic target in dry eye disease.J Ocul Pharmacol Ther. 2017; 33: 5-12Crossref PubMed Scopus (70) Google Scholar Here, antigen-presenting cells induce the differentiation of effector helper T cells (Th) that migrate through the efferent blood vessels to the ocular surface, where they release proinflammatory factors.17Stevenson W. Chauhan S.K. Dana R. Dry eye disease.Arch Ophthalmol. 2012; 130: 90Crossref PubMed Scopus (407) Google Scholar, 18Barabino S. Chen Y. Chauhan S. Dana R. Ocular surface immunity: homeostatic mechanisms and their disruption in dry eye disease.Prog Retin Eye Res. 2012; 31: 271-285Crossref PubMed Scopus (212) Google Scholar, 19Foulsham W. Coco G. Amouzegar A. Chauhan S.K. Dana R. When clarity is crucial: regulating ocular surface immunity.Trends Immunol. 2018; 39: 288-301Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar,22Stern M.E. Schaumburg C.S. Pflugfelder S.C. Dry eye as a mucosal autoimmune disease.Int Rev Immunol. 2013; 32: 19-41Crossref PubMed Scopus (241) Google Scholar Thus, the immunopathogenesis of DED can be understood as a self-perpetuating cycle, whereby a proinflammatory microenvironment engenders further infiltration of pathogenic immune cells and increases damage to the ocular surface.23Bron A.J. de Paiva C.S. Chauhan S.K. Bonini S. Gabison E.E. Jain S. Knop E. Markoulli M. Ogawa Y. Perez V. Uchino Y. Yokoi N. Zoukhri D. Sullivan D.A. TFOS DEWS II pathophysiology report.Ocul Surf. 2017; 15: 438-510Crossref PubMed Scopus (771) Google Scholar,24Yamaguchi T. Inflammatory response in dry eye.Invest Ophthalmol Vis Sci. 2018; 59: DES192Crossref PubMed Scopus (110) Google Scholar Th17 cells have emerged as key effector immune cells in DED,25Chauhan S.K. El Annan J. Ecoiffier T. Goyal S. Zhang Q. Saban D.R. Dana R. Autoimmunity in dry eye is due to resistance of Th17 to Treg suppression.J Immunol. 2009; 182: 1247-1252Crossref PubMed Scopus (225) Google Scholar, 26Chauhan S.K. Dana R. Role of Th17 cells in the immunopathogenesis of dry eye disease.Mucosal Immunol. 2009; 2: 375-376Crossref PubMed Scopus (79) Google Scholar, 27De Paiva C.S. Chotikavanich S. Pangelinan S.B. Pitcher J.D. Fang B. Zheng X. Ma P. Farley W.J. Siemasko K.F. Niederkorn J.Y. Stern M.E. Li D.-Q. Pflugfelder S.C. IL-17 disrupts corneal barrier following desiccating stress.Mucosal Immunol. 2009; 2: 243-253Crossref PubMed Scopus (301) Google Scholar mirroring their immunopathogenic role in autoimmune conditions such as uveitis/scleritis,28Amadi-Obi A. Yu C.-R. Liu X. Mahdi R.M. Clarke G.L. Nussenblatt R.B. Gery I. Lee Y.S. Egwuagu C.E. TH17 cells contribute to uveitis and scleritis and are expanded by IL-2 and inhibited by IL-27/STAT1.Nat Med. 2007; 13: 711-718Crossref PubMed Scopus (683) Google Scholar rheumatoid arthritis,29Hirota K. Yoshitomi H. Hashimoto M. Maeda S. Teradaira S. Sugimoto N. Yamaguchi T. Nomura T. Ito H. Nakamura T. Sakaguchi N. Sakaguchi S. Preferential recruitment of CCR6-expressing Th17 cells to inflamed joints via CCL20 in rheumatoid arthritis and its animal model.J Exp Med. 2007; 204: 2803-2812Crossref PubMed Scopus (705) Google Scholar inflammatory bowel disease,30Abraham C. Cho J. Interleukin-23/Th17 pathways and inflammatory bowel disease.Inflamm Bowel Dis. 2009; 15: 1090-1100Crossref PubMed Scopus (238) Google Scholar and multiple sclerosis.31Kebir H. Kreymborg K. Ifergan I. Dodelet-Devillers A. Cayrol R. Bernard M. Giuliani F. Arbour N. Becher B. Prat A. Human TH17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation.Nat Med. 2007; 13: 1173-1175Crossref PubMed Scopus (1213) Google Scholar Experimental desiccating stress–induced DED is a well-recognized preclinical model of ocular surface autoimmunity.25Chauhan S.K. El Annan J. Ecoiffier T. Goyal S. Zhang Q. Saban D.R. Dana R. Autoimmunity in dry eye is due to resistance of Th17 to Treg suppression.J Immunol. 2009; 182: 1247-1252Crossref PubMed Scopus (225) Google Scholar,32Stern M.E. Schaumburg C.S. Dana R. Calonge M. Niederkorn J.Y. Pflugfelder S.C. Autoimmunity at the ocular surface: pathogenesis and regulation.Mucosal Immunol. 2010; 3: 425-442Crossref PubMed Scopus (87) Google Scholar Memory Th17 cells (CD4+CD44hiIL-17A+) are critical mediators of DED chronicity, and are responsible for the heightened disease severity observed when mice are rechallenged with desiccating stress.33Chen Y. Chauhan S.K. Soo Lee H. Saban D.R. Dana R. Chronic dry eye disease is principally mediated by effector memory Th17 cells.Mucosal Immunol. 2014; 7: 38-45Crossref PubMed Scopus (71) Google Scholar In the experimental autoimmune encephalitis model, memory Th17 cells also have been implicated in promoting earlier onset and increased severity of clinical disease after rechallenge.9Haines C.J. Chen Y. Blumenschein W.M. Jain R. Chang C. Joyce-Shaikh B. Porth K. Boniface K. Mattson J. Basham B. Anderton S.M. McClanahan T.K. Sadekova S. Cua D.J. McGeachy M.J. Autoimmune memory T helper 17 cell function and expansion are dependent on interleukin-23.Cell Rep. 2013; 3: 1378-1388Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar However, the contribution of the memory Th17 cell population to age-associated ocular surface autoimmunity is not known. In the present study, we investigated the role of memory Th17 cells in DED pathogenesis in aged mice. Specifically, DED severity was evaluated in young (6- to 8-week–old) versus aged (12- to 14-month–old) mice during primary and secondary challenges. We quantified memory Th17 populations in young versus aged mice after exposure to desiccating stress, and evaluated memory Th17 cell depletion as a strategy to treat severe DED exacerbations in aged mice. Six- to 8-week–old (young) and 12- to 14-month–old (aged) female C57BL/6 mice from Charles River Laboratories (Wilmington, MA) were used in these experiments. Animals were housed in the Schepens Eye Research Institute animal vivarium. Animals were treated according to The Association for Research in Vision and Ophthalmology's Statement for the Use of Animals in Ophthalmic and Vision Research. Experiments were approved by the Schepens Eye Research Institute Animal Care and Use Committee. DED was induced as described previously.33Chen Y. Chauhan S.K. Soo Lee H. Saban D.R. Dana R. Chronic dry eye disease is principally mediated by effector memory Th17 cells.Mucosal Immunol. 2014; 7: 38-45Crossref PubMed Scopus (71) Google Scholar, 34Tan X. Chen Y. Foulsham W. Amouzegar A. Inomata T. Liu Y. Chauhan S.K. Dana R. The immunoregulatory role of corneal epithelium-derived thrombospondin-1 in dry eye disease.Ocul Surf. 2018; 16: 470-477Crossref PubMed Scopus (21) Google Scholar, 35Chen Y. Chauhan S.K. Tan X. Dana R. Interleukin-7 and -15 maintain pathogenic memory Th17 cells in autoimmunity.J Autoimmun. 2017; 77: 96-103Crossref PubMed Scopus (36) Google Scholar In brief, mice were housed for 14 days in a controlled environment chamber with continuous airflow of 15 L/min and relative humidity maintained at less than 20%. Naive control mice were housed in the standard vivarium. Corneal epitheliopathy was assessed by administering 1 μL of 1% fluorescein (Sigma-Aldrich Corp., St. Louis, MO) to the lateral conjunctival sac using a micropipette. After 3 minutes, punctate staining was evaluated using a slit-lamp biomicroscope under cobalt blue light, and the left eye of each mouse was scored according to the National Eye Institute grading scale.36Barabino S. Shen L. Chen L. Rashid S. Rolando M. Dana M.R. The controlled-environment chamber: a new mouse model of dry eye.Invest Ophthalmol Vis Sci. 2005; 46: 2766Crossref PubMed Scopus (142) Google Scholar The role of IL-15 in promoting the homeostatic proliferation of memory CD8+ T cells is well recognized, however, IL-15 signaling also has been shown to have a prominent nonredundant role in the survival and proliferation of memory CD4+ T cells.35Chen Y. Chauhan S.K. Tan X. Dana R. Interleukin-7 and -15 maintain pathogenic memory Th17 cells in autoimmunity.J Autoimmun. 2017; 77: 96-103Crossref PubMed Scopus (36) Google Scholar,37Lenz D.C. Kurz S.K. Lemmens E. Schoenberger S.P. Sprent J. Oldstone M.B.A. Homann D. IL-7 regulates basal homeostatic proliferation of antiviral CD4+T cell memory.Proc Natl Acad Sci U S A. 2004; 101: 9357-9362Crossref PubMed Scopus (164) Google Scholar, 38Purton J.F. Tan J.T. Rubinstein M.P. Kim D.M. Sprent J. Surh C.D. Antiviral CD4+ memory T cells are IL-15 dependent.J Exp Med. 2007; 204: 951-961Crossref PubMed Scopus (185) Google Scholar, 39Surh C.D. Sprent J. Homeostasis of naive and memory T cells.Immunity. 2008; 29: 848-862Abstract Full Text Full Text PDF PubMed Scopus (868) Google Scholar In this study, in vivo blockade with anti–IL-15 monoclonal antibody was used as a strategy to deplete the memory Th17 pool. Specifically, mice were administered either anti–IL-15 monoclonal antibody (25 μg in 100 μL sterile saline, Clone AIO.3; eBioscience, San Diego, CA) or control (100 μL sterile saline) once daily via i.p. injection for 7 days. Submandibular and draining lymph nodes were collected using jewelers forceps (Katena Products, Inc., Denville, NJ). Conjunctivae were harvested using jewelers forceps and Vannas scissors (Storz; Bausch & Lomb, Rochester, NY), before being digested in RPMI media (Lonza, Walkersville, MD) containing 2 mg/mL collagenase type IV (Sigma-Aldrich Corp.) and 2 mg/mL DNase I (Roche, Basel, Switzerland) for a duration of 45 minutes at a temperature of 37°C, as described previously.40Shukla S. Mittal S.K. Foulsham W. Elbasiony E. Singhania D. Sahu S.K. Chauhan S.K. Therapeutic efficacy of different routes of mesenchymal stem cell administration in corneal injury.Ocul Surf. 2019; 17 (ARVO Annua): 729-736Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar,41Mittal S.K. Foulsham W. Shukla S. Elbasiony E. Omoto M. Chauhan S.K. Mesenchymal stromal cells modulate corneal alloimmunity via secretion of hepatocyte growth factor.Stem Cells Transl Med. 2019; 8: 1030-1040Crossref PubMed Scopus (20) Google Scholar Subsequently, cells were passed through a 70-μm cell strainer (Falcon; Corning Life Sciences, Corning, NY). Single-cell suspensions were stained with the following fluorochrome-conjugated monoclonal antibodies: fluorescein isothiocyanate–conjugated anti-CD4 and peridinin-chlorophyll protein complex cyanine 5.5–conjugated anti-CD44 (BioLegend, San Diego, CA). For intracellular IL-17 staining, cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate and 500 ng/mL ionomycin (Sigma-Aldrich Corp.) for 6 hours at 37°C and 5% CO2 in the presence of GolgiStop (4 μL per 6-mL cell culture; BD Biosciences, San Jose, CA) to inhibit cytokine secretion. After this, cells were stained for phycoerythrin-conjugated anti–IL-17A (BioLegend). Control samples were stained with isotype-matched control antibodies. Stained cells were analyzed using a LSR II flow cytometer (BD Biosciences) and Summit software version 4.3 (Dako Colorado, Inc., Fort Collins, CO). Gating strategies for selecting CD4+ cells are shown in Supplemental Figure S1. Unpaired, two-tailed, t-tests and 2-way analysis of variance tests were used as appropriate to determine significance, which was set at P < 0.05. Data are presented as means ± SEM. Sample sizes were based on previous experimental studies of DED.33Chen Y. Chauhan S.K. Soo Lee H. Saban D.R. Dana R. Chronic dry eye disease is principally mediated by effector memory Th17 cells.Mucosal Immunol. 2014; 7: 38-45Crossref PubMed Scopus (71) Google Scholar,35Chen Y. Chauhan S.K. Tan X. Dana R. Interleukin-7 and -15 maintain pathogenic memory Th17 cells in autoimmunity.J Autoimmun. 2017; 77: 96-103Crossref PubMed Scopus (36) Google Scholar,42Dohlman T.H. Chauhan S.K. Kodati S. Hua J. Chen Y. Omoto M. Sadrai Z. Dana R. The CCR6/CCL20 axis mediates Th17 cell migration to the ocular surface in dry eye disease.Invest Ophthalmol Vis Sci. 2013; 54: 4081-4091Crossref PubMed Scopus (49) Google Scholar To evaluate corneal epitheliopathy after exposure to desiccating stress, corneal fluorescein staining was used, with representative images of different disease severities shown in Figure 1A. To examine the difference in clinical disease between young and aged mice on exposure to a dry environment, mice were housed in the controlled environment chamber for a period of 14 days, as previously described.34Tan X. Chen Y. Foulsham W. Amouzegar A. Inomata T. Liu Y. Chauhan S.K. Dana R. The immunoregulatory role of corneal epithelium-derived thrombospondin-1 in dry eye disease.Ocul Surf. 2018; 16: 470-477Crossref PubMed Scopus (21) Google Scholar,42Dohlman T.H. Chauhan S.K. Kodati S. Hua J. Chen Y. Omoto M. Sadrai Z. Dana R. The CCR6/CCL20 axis mediates Th17 cell migration to the ocular surface in dry eye disease.Invest Ophthalmol Vis Sci. 2013; 54: 4081-4091Crossref PubMed Scopus (49) Google Scholar,43Kodati S. Chauhan S.K. Chen Y. Dohlman T.H. Karimian P. Saban D. Dana R. CCR7 is critical for the induction and maintenance of Th17 immunity in dry eye disease.Invest Ophthalmol Vis Sci. 2014; 55: 5871-5877Crossref PubMed Scopus (39) Google Scholar Mice subsequently were returned to a standard vivarium for 21 days, before undergoing rechallenge with a further 14 days of desiccating stress (Figure 1B). No difference in corneal fluorescein staining score between young and aged mice was observed at baseline (young, 1.6; aged, 2.7), after 7 days of desiccation (young, 6.1; aged, 6.9), or after 14 days of desiccation (young, 8.5; aged, 9.9) (Figure 1C). However, upon rechallenge, substantially higher scores of corneal epitheliopathy were observed in aged mice compared with young mice (7 days: young, 10.0; aged, 13.7, P = 0.009; 14 days: young, 11.6; aged, 14.7, P < 0.001) (Figure 1C). Th17 cells have been identified as critical effector cells promoting inflammation in DED.25Chauhan S.K. El Annan J. Ecoiffier T. Goyal S. Zhang Q. Saban D.R. Dana R. Autoimmunity in dry eye is due to resistance of Th17 to Treg suppression.J Immunol. 2009; 182: 1247-1252Crossref PubMed Scopus (225) Google Scholar, 26Chauhan S.K. Dana R. Role of Th17 cells in the immunopathogenesis of dry eye disease.Mucosal Immunol. 2009; 2: 375-376Crossref PubMed Scopus (79) Google Scholar, 27De Paiva C.S. Chotikavanich S. Pangelinan S.B. Pitcher J.D. Fang B. Zheng X. Ma P. Farley W.J. Siemasko K.F. Niederkorn J.Y. Stern M.E. Li D.-Q. Pflugfelder S.C. IL-17 disrupts corneal barrier following desiccating stress.Mucosal Immunol. 2009; 2: 243-253Crossref PubMed Scopus (301) Google Scholar In view of our data showing severe corneal epitheliopathy in aged compared with young mice after rechallenge, we next sought to determine the frequencies of Th17 cells in the respective groups. Aged rechallenged mice were observed to have substantially higher lymph node Th17 frequencies compared with aged naive mice (4.2% ± 0.3% versus 1.5% ± 0.3%, respectively; P = 0.004) (Figure 2A). Young rechallenged mice also had a tendency for higher Th17 frequencies in the draining lymph nodes relative to naive mice (1.4% ± 0.5% versus 0.7% ± 0.3%, respectively; P = 0.248). Notably, however, aged mice showed a 3.9-fold greater increase in lymph node Th17 frequencies relative to young mice (2.7% versus 0.7%, respectively). A two-way analysis of variance was conducted on the influence of age and desiccating stress rechallenge on the corneal fluorescein staining score. All effects were statistically significant at the 0.05 significance level. The main effect for age yielded an F ratio of 26.5 (P < 0.001), indicating a significant difference between young and aged mice. The main effect for desiccating stress rechallenge yielded an F ratio of 9.7 (P < 0.001), showing a significant difference between naive and rechallenged mice. Finally, the interaction effect was significant (P = 0.022). After rechallenge, Th17 frequencies in the conjunctivae of young mice were observed to increase to 6.6% ± 1.1% relative to 2.6% ± 0.6% in naive mice (P = 0.011) (Figure 2B). Analysis of the conjunctivae of aged mice showed a 2.2-fold greater increase in Th17 infiltration (aged, 8.9%; young, 4.0%), with frequencies of 11.3% ± 1.2% detected in aged mice after rechallenge and 2.4% ± 0.4% in naive mice (P < 0.001). A two-way analysis of variance was performed, with all effects statistically significant at the 0.05 significance level. The main effect for age showed an F ratio of 7.6 (P = 0.014), showing a significant difference between young and aged mice. The main effect for desiccating stress rechallenge yielded an F ratio of 43.8 (P < 0.001), showing a significant difference between naive and rechallenged mice. The interaction effect also was significant (P = 0.035). Memory Th17 cells (CD4+CD44highIL-17+) are critical mediators of the low-grade inflammation observed in chronic DED.33Chen Y. Chauhan S.K. Soo Lee H. Saban D.R. Dana R. Chronic dry eye disease is principally mediated by effector memory Th17 cells.Mucosal Immunol. 2014; 7: 38-45Crossref PubMed Scopus (71) Google Scholar,35Chen Y. Chauhan S.K. Tan X. Dana R. Interleukin-7 and -15 maintain pathogenic memory Th17 cells in autoimmunity.J Autoimmun. 2017; 77: 96-103Crossref PubMed Scopus (36) Google Scholar To delineate the contribution of memory Th17 cells to the severe exacerbations of DED observed in aged mice, an experiment was designed in which young and aged mice would be treated systemically with anti–IL-15 monoclonal antibody (mAb) after primary challenge with desiccating stress to reduce the memory Th17 population. After 2 weeks in the controlled environment chamber, mice were returned to a standard vivarium for 3 weeks (Figure 3A). During the first of these 3 weeks in the standard vivarium, mice received daily i.p. injections with anti–IL-15 mAb. After these 3 weeks, mice were returned to the controlled environment for a 2-week rechallenge. Mice were sacrificed and their tissues were harvested for immune analysis at two time points: either before rechallenge or after rechallenge. Tissue analysis before rechallenge showed a significant increase in memory Th17 frequencies in both young and aged saline-treated animals that had been exposed previously to desiccating stress relative to naive mice (P = 0.042 and P = 0.017, respectively) (Figure 3, B and C). However, the increase in memory Th17 frequencies in aged mice was substantially greater (approximately threefold) compared with young mice. Treatment with anti–IL-15 abrogated the increase in memory Th17 cells in both young and aged mice (P = 0.032 and P = 0.010, respectively) (Figure 3, B and C). A two-way analysis of variance was performed, with all effects statistically significant at the 0.05 significance level. The main effect for age showed an F ratio of 26.4 (P < 0.001), showing a significant difference between young and aged mice. The main effect for desiccating stress rechallenge yielded an F ratio of 16.7 (P < 0.001), showing a significant difference between naive rechallenged and anti–IL-15–treated mice. Finally, the interaction effect was significant (P = 0.034). Having observed the reduction in memory Th17 cell frequencies in aged animals resulting from treatment with anti–IL-15 mAb, we sought to determine the effect of memory Th17 cell depletion on DED severity. Mice were exposed to desiccating stress and treatment with anti–IL-15 mAb as shown in Figure 3A. Evaluation of corneal fluorescein staining scores showed that treatment with anti–IL-15 mAb resulted in a significant reduction in DED severity in aged mice at day 7 (26%; P = 0.007) and at day 14 (29%; P < 0.001) after rechallenge (Figure 4A). Mice were sacrificed at 2 weeks after rechallenge, and flow cytometric analysis of single-cell suspensions derived from conjunctivae showed decreased effector Th17 cell infiltration in the anti–IL-15 mAb–treated mice relative to the saline control animals (5.1% compared with 10.1%, respectively; P = 0.016) (Figure 4, B and C). The aged immune system is susceptible to autoimmunity, and many autoimmune conditions preferentially occur later in life.3Goronzy J.J. Weyand C.M. Immune aging and autoimmunity.Cell Mol Life Sci. 2012; 69: 1615-1623Crossref PubMed Scopus (152) Google Scholar Despite extensive investigations into the role of effector Th17 cells in autoimmunity, little is known about the contribution of memory Th17 cells to age-related autoimmune conditions. In this study, a murine model of ocular surface autoimmunity was used to show the following: i) aged mice develop more severe exacerbations of DED relative to young mice; ii) after primary exposure to desiccating stress, aged mice maintain an amplified memory Th17 pool; and iii) depletion of the memory Th17 population with anti–IL-15 mAb abrogates both the increased clinical disease and up-regulated tissue infiltration of effector Th17 cells observed in
Referência(s)