Browsing by Author "Zhang, Jennifer"

As human skin ages, its ability to both repair wounds and protect them from infection declines. Several factors play major roles in this, including thinning of epidermis and loss of collagen leading to skin fragility, as well as the decline of innate immune function, though the latter has been less distinctly linked to skin aging. Given the rising aging population globally, understanding how the skin responds to injury across the life spectrum is increasingly important. This work attempts to understand specifically how innate antiviral immunity of the skin is downregulated in age. To do so, we leveraged a discovery where we determined that aging skin has a differential circadian clock, a known immunological regulator, compared to that of younger skin. The circadian rhythm is a biological clock that uses a transcriptional-translational feedback loop to set up patterns of activity throughout the body. This loop uses positive transcription factors BMAL1 and CLOCK, which set up their own repressors, including the PER and CRY family of proteins. This rhythm also influences biological functions throughout the body. In the scope of this work, we became interested in the fact that circadian rhythms were found to influence epithelial repair in injury, antiviral immunity and interferon stimulated genes. To begin our study, we asked whether the known repair and immune dysfunctions of aging skin could be possibly tied to a dysfunctional circadian rhythm. Using qRT-PCR, we found that aging murine skin has a decreased circadian transcription when compared to that of younger skin. Such a phenotype was replicated by human keratinocyte studies using serial passaging as an aging surrogate. Having determined that aging does indeed play a role in regulating cutaneous circadian rhythms, we set out to determine what immune mechanisms of the skin are regulated by this aging-circadian axis. Specifically, we tested circadian regulation of antiviral proteins. Antiviral proteins of distinct families and functions all protect the skin from pathogen invasion. Prior work by our lab had shown that antiviral proteins were induced by skin wounding in a pathway dependent on the cytokine IL-27. Using approaches including qRT-PCR, flow cytometry, and immunofluorescence, we determined that aging skin wounds not only have an attenuated antiviral protein response, but also contained reduced numbers of CD301b+ immune cells that produce IL-27. These distinct immune deficiencies lead to an unprotected skin wound microenvironment in aging skin. However, little is understood about the molecular mechanisms responsible for the antiviral immune deficiencies in the aging skin. To address this, we began by probing publicly available datasets, where we found that the expression of antiviral proteins had 24-hour rhythms of expression in murine skin. Similarly, we found that that rhythmic expression of antiviral proteins occurs in human keratinocytes that were synchronized in a circadian fashion. Additional support for a direct line of circadian regulation of antiviral proteins came from circadian siRNA studies, where we knocked down expression of circadian gene CLOCK and saw an associated downregulation in antiviral proteins within human keratinocytes. Further, we demonstrate via qRT-PCR that murine skin harvested at different time points have different antiviral protein mRNA levels. Subsequent computational analysis showed that Bmal1-/- murine skin is deficient in antiviral protein expression, establishing a direct link between circadian factors and antiviral proteins. In order to better understand the effect of circadian rhythms on wound immune responses, we made use of a number of experimental models, including both Bmal1-/- and ClockΔ19 mutant mice, as well as wild type animals. We wounded wild type animals at distinct time-of-day, and found that the level of antiviral proteins display time-of-day responses, peaking at 8pm. Using circadian mutant mice, we found that these animals have attenuated wound responses with respect to antiviral protein induction; specifically, wounded ClockΔ19 mice do not produce antiviral proteins to the same extent as wild type mice. We were able to tie this directly to IL-27 signaling in two distinct manners. Firstly, using flow cytometry, we found that the CD301b+ cells that produce IL-27 in response to wounding are reduced in number in circadian mutant mouse skin, and moreover, produce less IL-27 as measured by median fluorescence intensity. To determine the role of IL-27 in the time-of-day response of wound-induced antiviral protein expression, we wounded IL-27fl/fl-LysM-Cre mice at two distinct times-of-day and measured antiviral protein production. These mice lack IL-27 production from myeloid cell lineages, including CD301b+ cells. We found that loss of IL-27 diminished time-of-day differential expression of antiviral proteins in wounds, further suggesting that the link between circadian rhythms and antiviral proteins was in fact in part mediated by IL-27. To further our understanding of the cytokine milieu of circadian wounds, we also wounded mice that lacked Type I interferon receptor (IFNAR1) expression and found that loss of Type I interferon signaling also blunted time-of-day antiviral protein responses. These data support a role of both interferons and IL-27 in circadian antiviral protein induction. In order to provide a functional aspect to these findings, we infected wildtype and circadian disrupted keratinocytes and human skin with Herpes Simplex Virus Type I (HSV1). We measured HSV expression in the skin by both immunofluorescence and visual characterization as well by qPCR for viral component UL29. We found that circadian disruption of either BMAL1 or CLOCK sensitizes keratinocytes to HSV1 infection in vitro. On the other side of this spectrum, we questioned whether circadian enhancing drugs, including the compounds SR8278 and nobiletin, can activate circadian rhythms in skin cells and improve skin defense against HSV1 infection. Using a BMAL1:Luciferase reporter, we characterized both drugs as having a circadian augmenting effect in keratinocytes. Upon infection with HSV1, both SR8278 and nobiletin protected human skin from viral spread. Further, we found that SR8278’s antiviral effect was predicated on circadian activity, as BMAL1 and CLOCK siRNA knockdown in keratinocytes lessened the drugs effect. To determine mechanism of circadian drug’s antiviral activity, we tested whether our circadian drugs activate canonical antiviral signaling pathways, such as OAS and IFITM. We found that via qPCR, circadian drugs require the presence of these proteins to fight virus effectively. As a study of clinical relevance, we evaluated the role of acyclovir treatments alongside our circadian drugs. We found that circadian drugs SR8278 and nobiletin did not synergize their effects with acyclovir at a variety of doses tested. We found that acyclovir, as expected, broadly suppressed HSV1 activity at even low doses in keratinocytes, an effect that circadian augmentation was unable to potentiate. This could be due to a number of factors, including dosage optimization and viral susceptibility to drug. However, given the rise of acyclovir resistant HSV, our novel approach may be clinically viable. In particular, we believe this may be a viable treatment platform for aging skin infections; to this end, we tested the ability of SR8278 to suppress HSV1 infection in the skin of mice over a year of age. SR8278 significantly reduced viral spread in this model, suggesting that circadian augmentation may be a useful clinical adjunct in aging skin infections. To determine if these findings were applicable to other non-herpes family viruses that infect the skin, we turned to a model of West Nile Virus infection. West Nile Virus is a mosquito borne illness with increasing range and infection number in the United States. Moreover, it is inoculated through the skin before causing neurological infection, a pathway similar to herpes viruses. Also similar to herpes viruses, West Nile virus is a far more pressing clinical issue in aging populations, who fare worse with this viral infection. Most importantly, there are no currently specific treatments for West Nile Virus infections. Using our HSV infection data as a starting point, we found that circadian drug treatments suppressed West Nile Virus levels in infected keratinocytes. Other work conducted over the course of this PhD encompassed aspects of both circadian and IL-27 signaling in the skin. Using human keratinocytes, we endeavored to understand what environmental factors could drive altered circadian rhythms in the skin, for either elderly or younger tissue. While dogmatically, circadian rhythms are patterned from the brain to the whole body, we built on recent work showing a light dependent murine cutaneous clock by showing that mock sunlight can alter circadian expression in human keratinocytes, without other stimuli present. Further study is needed to understand how our skin’s clock responds to sun mechanistically. Overall, my work over the course of this PhD has established a link between aging, circadian rhythms, and antiviral immunity, and underpinned the important role of the cytokine IL-27 and type I interferon on cutaneous wound responses to a variety of pathogens. This work will provide possible therapeutic avenues, particularly for aging skin, in how to address skin wound care in safe, biologically relevant ways via circadian rhythm exploitation.

Ubiquitination is a post-translational modification that mediates protein stability and function, and it is implicated in almost every aspect of cellular activities. UBE2N is an E2 conjugating enzyme that mediates Lys-63 (K63) polyubiquitination. It plays essential roles in signal transduction and DNA repair mechanisms. In this thesis, we utilized two conditional mouse models to investigate the role of UBE2N in the skin: Rosa26CreER.Ube2nfl/fl and Krt5CreER.Ube2nfl/fl. We found that the conditional deletion of Ube2n in adult skin resulted in inflammatory skin defects. Using histology, single-cell RNA-sequencing, RT-qPCR, flow cytometry, immunofluorescent staining, and western blotting, we learned that the inflammatory skin accompanied epidermal and dermal thickening, parakeratosis, and increased immune cell infiltration. The skin expressed a pro-inflammatory gene signature including elevated levels of cytokines such as Il1 and Il24 and chemokines such as Cxcl1 and Cxcl2. The immune infiltration was primarily of myeloid origin including neutrophils and M1-like proinflammatory macrophages. Using the basal keratinocyte-specific Krt5CreER.Ube2nfl/fl transgenic mouse model, we also learned that loss of UBE2N in keratinocytes is sufficient to induce inflammatory skin defects that was observed in the Rosa26CreER.Ube2nfl/fl mice. The Ube2n knockout (KO) keratinocytes also showed increased levels of pro-inflammatory cytokine and myeloid cell chemokine genes. Consistently, the Ube2n KO skin showed infiltration of myeloid cells. Furthermore, we discovered that genes in the IL-1 signaling pathway are highly upregulated in both the Rosa26CreER.Ube2nfl/fl and the Krt5CreER.Ube2nfl/fl. Of them, interleukin-1 receptor-associated kinase 1 (IRAK1) was activated in the Ube2n KO skin. To test the hypothesis that the loss of Ube2n induced inflammatory phenotypes are mediated by IL-1 signaling pathway, we treated Krt5CreER.Ube2nfl/fl mice with an IRAK1 and 4 (IRAK1/4)-specific pharmacological inhibitor. The IRAK1/4 inhibitor reduced the level of inflammatory phenotypes of the Ube2n KO skin. This suggested that IRAK1/4 signaling pathway plays an important role in mediating the inflammation caused by loss of Ube2n. Many chronic inflammatory skin disorders are accompanied by cutaneous dysbiosis. Since loss of UBE2N led to inflammatory skin lesions with disrupted epidermis and increased pro-inflammatory immune signature, we also investigated whether UBE2N plays a role in cutaneous microbial regulation. In order to assess the cutaneous microbiome of the Ube2n KO skin, we performed an amplicon sequencing analysis of the V4 region of 16S rRNA gene from skin swabs of the and the control mice. The sequencing data showed that there was a dramatic shift in microbial population: an increased abundance of Corynebacterium in the inflamed Ube2n KO skin. In summary, our investigation of the role of UBE2N and its function in the skin revealed that it is critical for maintaining epidermal homeostasis and suppression of skin inflammation. We also learned that UBE2N loss-of-function-mediated skin inflammation leads to dysbiosis. This suggests that the restoration of UBE2N signaling and/or IRAK1/4 inhibition could be potential therapeutic strategies for inflammatory skin disorders and controlling healthy skin microbiome.