Mechanisms of 2,4-Dinitrochlorobenzene-induced acute inflammation in Human Papillomavirus type 16 E7 oncoprotein expressing skin

Tran, Le Son (2015). Mechanisms of 2,4-Dinitrochlorobenzene-induced acute inflammation in Human Papillomavirus type 16 E7 oncoprotein expressing skin PhD Thesis, UQ Diamantina Institute, The University of Queensland. doi:10.14264/uql.2015.311

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Author Tran, Le Son
Thesis Title Mechanisms of 2,4-Dinitrochlorobenzene-induced acute inflammation in Human Papillomavirus type 16 E7 oncoprotein expressing skin
School, Centre or Institute UQ Diamantina Institute
Institution The University of Queensland
DOI 10.14264/uql.2015.311
Publication date 2015-01-30
Thesis type PhD Thesis
Open Access Status Other
Supervisor Ian Frazer
Stephen Mattarollo
Deepak Mittal
Total pages 162
Language eng
Subjects 1107 Immunology
1112 Oncology and Carcinogenesis
Formatted abstract
Persistent infection with oncogenic human papillomaviruses (HPV), particularly HPV16, is associated with selective expression of two virally encoded proteins (E6 and E7). One action of HPV16.E7 protein is to subvert the innate immune system through the down-regulation of IFNγ pathways, modulation of antigen presentation, and suppression of Toll-like receptor 9 protein.

K14.E7 transgenic mice, which express HPV16.E7 oncoprotein within basal keratinocytes under the control of the keratin 14 transcriptional promoter, have been extensively used as a model of HPV oncoprotein–induced immune suppression associated with human squamous cancers, in which only the E6 and E7 genes of the papillomavirus are expressed. It has previously been shown that skin grafts expressing HPV16.E7 oncoprotein are not spontaneously rejected when transplanted onto syngeneic animals, but they are rejected when certain components of the innate immune system are unavailable, confirming that the expression of HPV16.E7 in the epithelium results in the establishment of a local suppressive environment and the subversion of antigen-specific T cells. Therefore, successful strategies targeting HPV-associated cancer need to circumvent or disrupt the local suppressive environment.

Topical immunotherapy with immunostimulatory agents has been used clinically to treat cancerous lesions including squamous cell and basal cell carcinoma in immunocompetent and immunosuppressed patients. Topical application of 2,4-dinitrochlorobenzene (DNCB) is an effective therapy for condylomata acuminata caused by HPV infection. DNCB induced the complete clearance of HPV-associated warts in 13/15 patients. However, the use of DNCB for treatment has been discouraged because of its mutagenic potential. The aim of my doctoral research is to understand the mechanism of DNCB-induced inflammation in K14.E7 transgenic mice which has been poorly understood. I hypothesized that understanding how induction of a vigorous acute inflammatory response by DNCB can break the local immune-suppressive environment and restore the effector function of adaptive immunity might lead to more acceptable treatments for persisting HPV infection. In support of this hypothesis, the present study showed that DNCB application along with E7 specific immunization therapy was capable of inducing HPV16.E7 skin graft rejection.

Arginase, which metabolizes l-arginine to N-ornithine and urea, has been identified as a crucial regulator of inflammation. As inflammation decides the fate of HPV infection and arginase is an important regulator of inflammation and immunity, I investigated the interaction between DNCB-induced inflammation and HPV16.E7 protein in the induction of arginase in K14.E7 transgenic mice. The first part of this study showed that topical DNCB application to skin expressing HPV16.E7 as a transgene induces a hyperinflammatory response, which is not seen in nontransgenic control animals. The E7-associated inflammatory response was characterized by enhanced expression of Th2 cytokines and increased infiltration of CD11b+Gr1intF4/80+Ly6ChiLy6Glow myeloid cells, producing arginase-1. Inhibition of arginase with an arginase-specific inhibitor, N(omega)-hydroxy-nor-L-arginine, ameliorated the DNCB-induced inflammatory response.

Furthermore, Th2 cytokines but not Th1 cytokines are induced in K14.E7 skin and these cytokines have been well-known as potent inducers of arginase-1 production in macrophages. In the second part of my study, I demonstrated that neutralization of these cytokines did not change the level of arginase activity in bone marrow-derived macrophages cultured in DNCB treated K14.E7 skin explant supernatant, suggesting that IL-4 and IL-10 might not be necessary for the induction of arginase activity in DNCB treated K14.E7 skin. Interestingly, IL-17A, an important proinflammatory cytokine, was also induced in K14.E7 mice but not in wild-type nontransgenic mice and CD11b+F4/80+ but not lymphocytes are the major producer of IL-17A in DNCB treated K14.E7 skin. Importantly, IL-17A deficient K14.E7 mice express diminished arginase activity and develop an alleviated inflammatory response to DNCB. Furthermore, I demonstrated that the induction of arginase-1 is independent of adaptive immunity. Thus, IL-17A produced by macrophage is responsible for enhanced arginase-1 expression in these cells.

Taken together, my study suggests that HPV16.E7 protein enhances DNCB associated-production of IL-17A which mediates arginase-1 production by inflammatory monocytes (Macrophages) and consequent inflammatory cellular infiltration of the skin. Thus, these findings provide insights into the pro-inflammatory roles of IL-17A and arginase-1 axis in HPV16.E7 expressing skin in response to immunostimulation by DNCB, and imply a potential immune-based therapy strategy to break the tolerance to HPV persistent infection by modulating IL-17A/arginase-1 axis.
Keyword HPV 16 E7
Cutaneous inflammation
Cervical cancer
Squamous cancer

Document type: Thesis
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Created: Fri, 16 Jan 2015, 16:15:00 EST by Mr Le Son Tran on behalf of Scholarly Communication and Digitisation Service