The Effect Of Aging On Development And Function Of Epidermal Langerhans Cells In Mice

Immunosenescence is a result of progressive and gradual decline in immune systemfunction with advancing age. The effect on the immune system due to aging involves bothhumoral and cell mediated immunity. Associated with a generalized impairment of immunefunction, it is known that aged populations are increasingly susceptible to cutaneous viral andfungal infections as well as to skin cancers that are accompanied with immunologicaldeficiencies. Langerhans cells (LCs), belonging to the dendritic cell (DC) family, which areprofessional antigen-presenting cells (APCs) that are important in the initial priming of na veT cells and act as a bridge to link innate immunity and adaptive immunity, act as sentinels toplay key roles in the skin immune responses. Langerin is the main molecular component ofBirbeck granules and plays a substantial role in antigen uptake. They capture and processexogenous antigens and migrate to the skin LNs where they activate T lymphocytes and thenplay an important role in cutaneous viral and fungal infections as well as to skin cancers. Withthe further study of aging and cutaneous immune, the effect of aging on the development andfunction of LC is more highly valued.In a well-defined classical TGF-β linear signaling pathway, once activated, TGF-β1signals through its two cell surface receptors, TGF-β receptor1(TβR1) and TGF-β receptor2(TβR2), leading to Smad-mediated transcriptional regulation. There are eight Smads: Smad1to Smad8. Smad2and Smad3are activated through carboxy-terminal phosphorylation byTGFβ1. These receptor-activated Smads (R-Smads) are released from the receptor complex toform a heterotrimeric complex with a common Smad4, and translocate into the nucleus toregulate the transcription of target genes. In addition, TGF-β1also can activate other signalingcascades, including MAPK pathways. Unlike mice with the conventional disruptions ofSmad2and Smad4that are lethal, mice with Smad3deficiency are viable and can survive. However, Smad3knockout (KO) mice develop progressive diseases, including leucocytosisand massive inflammation. The loss of Smad3results in multiple cell defects, including Tcells, neutriphils and macrophage. However, if TGFβ/Smad3signaling pathway is involved inthe ontogeny and homeostasis of epidermal LCs which is not clear so far.Immunosenescence is a result of progressive and gradual decline in immune systemfunction with advancing age. The effect on the immune system due to aging involves bothhumoral and cell mediated immunity. Defects in cellular immunity include a decrease inabsolute numbers of na ve T cells and alterations of T cell function. Recent studies haveshown that aging related defects in the immune system are not restricted to adaptive immunitybut can also extend to the innate immune response. There are many reports suggesting thatdifferent cellular components of the innate immune system such as neutrophils, natural killer(NK) cells, NKT, and macrophages, which are all important first lines of defense againstbacterial and parasitic infections, are defective in aged mice and human. DCs are professionalantigen-presenting cells (APCs) that are important in the initial priming of na ve T cells andact as a bridge to link innate immunity and adaptive immunity. There is also some controversyregarding the capacity of aged DCs to stimulated T cells. Epidermal LCs are skin-residentDCs with a life cycle distinct from other types of DCs. However, the precise effects of agingon LCs are still not well understood.Aging is a multifactorial process where deterioration of organism functions is driven bydistinct genetically encoded genes. To better understand the genetic component of aging,many studies have addressed the distinct gene and protein expression profiles in differentaging cells. microRNAs (miRNAs), small non-coding RNAs, have recently emerged as keyregulators of gene expression through mRNA degradation, and/or translation inhibition. Theribonuclease III enzyme Dicer is required for the processing of mature miRNAs. Therefore,deletion of Dicer provides a good genetic tool to test the relevance of miRNAs in mammaliandevelopment. Interestingly, deletion of Dicer specifically in DCs only affects the homeostasisand function of LCs, but not other types of DCs. This suggests that miRNAs are required fornormal LCs development and function. However, it is unclear whether aging affects miRNAgene expression in LCs, which may contribute to aging-mediated LC development andfunction. In this study, we will do experiments with Smad3-deficient mice model and aging micemodel to address the signal pathway for the development of LC and the role of aging in thedevelopment and function of epidermal LC by virtue of modern immunological techniques.Further study will confirm miRNA gene expression in LCs, which may contribute toaging-mediated LC development and function. The study includes three parts:1. The signal pathway for the development of epidermal LC: First we used Smad3KOmice to directly test if TGFβ/Smad3signaling pathway is involved in the ontogeny andhomeostasis of epidermal LCs. The data showed that Smad3is not required in the TGF-βsignal pathway for oncogeny and homeostasis of epidermis LC by by flow cytometry andimmunohistological staining analysis.Upon activation by various stimuli, immature LCs residing in epidermis collect antigenand increase their MHC II and costimulatory molecules, including CD80and CD86, and thenmigrate to T cells areas of draining lymph nodes, leading to immune responses. after60hculture, the relative CD80, CD86, and MHCII expression levels on LCs were significantlyincreased in Smad3KO and WT mice compared to unstimulation condition, but there was nosignificant difference between Smad3KO and WT mice (P>0.05). Thus, the lack of Smad3signaling does not affect the immature state of LCs as well as LC maturation. Due to theirphysical location, LCs acquire and process antigens, we further evaluate the role of Smad3inantigen phagocytic function of LCs. The result showed that lack of Smad3signal pathwaydoes not affect LC phagocytosis.In summary, lack of Smad3surprisingly does not significantly interrupt the developmentand immature state of epidermal LCs, and Smad3-deficient LCs have normal maturation andphagocytosis. Our data suggest that Smad3is not required in the TGF-β signal pathway forontogeny, homeostasis, and function of epidermal LCs.2. The effect of aging on the development and function of LC: Langerhans cells (LCs),belonging to the dendritic cell (DC) family, which are professional antigen-presenting cells(APCs) that are important in the initial priming of na ve T cells. With the further study ofaging and cutaneous immune, the effect of aging on the development and function of LC ismore highly valued. we examined the frequency of skin LCs at different time points. Thefrequency of epidermal LCs (Langerin+CD45.2+) was1.60±0.09%in <6months old mice and was significantly reduced to1.32±0.02%in12months old mice and1.13±0.10%in18months old mice. There was no significant difference between12months and18months oldmice. In consistence with reduced epidermal LCs, the proportion of Langerin+EpCAM+cellsin the gated MHCII+CD8-cells of LN, which represents the migrant epidermal LCs,decreased2-3-fold in12and18months old mice compared with6months old mice.Interestingly, the frequency of dendritic epidermal T cells (DETC) was comparable between6and12month old mice but significantly decreased at18months.The markedly reduced frequency of LCs in aged mice could be explained by either adecrease in the LC proliferation, increased LC apoptosis, or by a decrease in the number ofLCs progenitors. To study the first possibility, we preformed anti-Ki67antibody staining as away of measuring the kinetics of LCs turnover. We found no difference in the lifespan of LCsin aged and young mice. Furthmore LCs from aged mice exhibited normal rates of apoptosisas assessed by Annexin V and7-AAD staining compared to young LCs. This result furtherconfirms the previous observation by Sprecher et al., suggesting that the decreased LCsdensity in aged mice could result from a deficiency in local LCs progenitors.The initiation, execution, and sustainment of T cell immunity by DCs require cell-cellcontact mediated by costimulatory molecules on the DCs. The frequencies of maturationmarkers and expression levels of CD86and MHCII based on MFI were reduced on aged LCsThus, aged LCs have a lower ability to up-regulate MHCII and co-stimulatory moleculesupon in vitro maturation. LCs act as sentinels for contact with various environmental antigens.They capture and process exogenous antigens and migrate to the skin LNs where they activateT lymphocytes. Therefore we investigated whether aging can affect LC phagocytosis andmigration. The result showed that LCs from aged mice had increased phagocytic capacitycompared to that from young mice based on the ratio of FITC-positive or mean fluorescenceintensity expression levels. Morever, consistent with increased antigen uptake, we foundincreased Langerin expression in aged LCs. However, the migration ability of aged LCs didnot alter compared to young mice.LCs play a key role in mediating antigen-specfic T cell proliferation. The data showedthat aged LCs-mediated CD8+OT-I cell and CD4+OT-II T cell proliferation weredramatically reduced compared to young LCs, Thus, our data further suggest the defective LC-mediated T cell activation in aged mice.Our results demonstrate that the frequency and maturation of epidermal LCs werereduced in a stepwise fashion in aged mice and aged LCs exhibited a reduced ability tostimulate T cell proliferation. However, we need to further investigate the precise reasons.3. miRNAs expression and aged-LC function: previous studies suggested that miRNAswere required for normal LCs development and function. However, it is unclear whetheraging affects miRNA gene expression in LCs, which may contribute to aging-mediated LCdevelopment and function. In the current study, we systemically investigated the developmentand function of LCs during aging using C57BL/6J mice and performed miRNA expressionprofiles of LCs between aged and young mice. Using global normalization, we have identified53age-regulated miRNAs in LCs (fold change>3), including40downregulated miRNAsand13upregulated miRNAs.Based on the miRNAs potentially linked to LCs development and function, we havefurther confirmed that miR-709, miR-449and miR-9were upregualated in aging, whilemiR-200c and miR-10a were downregulated in aging by using single TaqMan RT-PCRassays.To explore the potential targets and pathways that are regulated by aging-associatedmiRNAs in LCs, we used the Target Scan Mouse6.2database to predict miRNA targetsrelated to LC homeostasis and function. As shown in Table1, Gfi1, IRF8, RunX3, C/EBP,TGFβRII, TGFβ1, AhR, RANKL, Langerin, and CSF1R are shown to regulate thehomeostasis, development and function of epidermal LCs, and are predictive targets byage-associated miRNAs.Above results demonstrate that Smad3is not required in the TGF-β signal pathway forontogeny, homeostasis, and function of epidermal LCs. Morever, the frequency andmaturation of epidermal LCs were reduced in a stepwise fashion in aged mice and aged LCsexhibited a reduced ability to stimulate T cell proliferation. Most important of all,genome-wide assessment of miRNA expression in epidermal LCs revealed a distinctaging-specific miRNA gene expression profile.In this study, we address the signal pathway for the development of LC and the role ofaging in the development and function of epidermal LC by virtue of modern immunological techniques. Further study will confirm miRNA gene expression in LCs, which may contributeto aging-mediated LC development and function.