Lack of Type 2 Innate Lymphoid Cells Promotes a Type I-Driven Enhanced Immune Response in Contact Hypersensitivity.

Allergic contact dermatitis and its animal model, contact hypersensitivity, are T-cell-mediated inflammatory skin diseases that require activation of the innate immune system. Here we investigate the role of innate lymphoid cells (ILCs) during the elicitation phase of 2,4,6-trinitrochlorobenzene-induced contact hypersensitivity using EomesGfp/+ x Rorc(γt)-CreTg x Rosa26RYfp/+ reporter mice. Ear swelling responses, cutaneous ILC numbers, and cytokine production were determined at different time points. Functional analyses were performed in a CD90.1/.2 congenic adoptive transfer model that allowed selective antibody-mediated depletion of ILCs before hapten challenge, and in Rorasg/floxIl7rCre/+ mice, which lack ILC2. Hapten challenge induced early increases of natural killer cells in skin and ear draining lymph nodes corresponding to the peak ear swelling response. In contrast, ILC1, 2, and 3 showed a delayed increase in numbers corresponding to the contact hypersensitivity resolution phase. Hapten challenge induced increased marker cytokines in all ILC subtypes and an activated phenotype in ILC2. Depletion of all ILC resulted in a significantly enhanced ear swelling response. Similarly, ILC2-deficient mice (Rorasg/floxIl7rCre/+) displayed increased ear swelling responses on hapten challenge, suggesting that ILC2 act as negative regulators in the type 1-dominated immune response of contact hypersensitivity.

Identification and Functional Characterization of Phosphorylation Sites of the Human Papillomavirus 31 E8

The papillomavirus E2 protein regulates transcription, replication, and nuclear retention of viral genomes. Phosphorylation of E2 in the hinge region has been suggested to modulate protein stability, DNA-binding activity, and chromosomal attachment. The papillomavirus E8^E2 protein shares the hinge domain with E2 and acts as a repressor of viral replication. Mass spectrometry analyses of human papillomavirus 31 (HPV31) E8^E2 and E2 proteins identify phosphorylated S78, S81, and S100 in E8^E2 and S266 and S269 in E2 in their hinge regions. Phos-tag analyses of wild-type and mutant proteins indicate that S78 is a major phosphorylation site in E8^E2, but the corresponding S266 in E2 is not. Phosphorylation at S78 regulates E8^E2's repression activity of reporter constructs, whereas the corresponding E2 mutants do not display a phenotype. Phosphorylation at S78 does not alter E8^E2's protein stability, nuclear localization, or binding to DNA or to cellular NCoR/SMRT complexes. Surprisingly, in the context of HPV31 genomes, mutation of E8^E2 S78 does not modulate viral replication or transcription in undifferentiated or differentiated cells. However, comparative transcriptome analyses of differentiated HPV31 E8^E2 S78A and S78E cell lines reveal that the expression of a small number of cellular genes is changed. Validation experiments suggest that the transcription of the cellular LYPD2 gene is altered in a phospho-S78 E8^E2-dependent manner. In summary, our data suggest that phosphorylation of S78 in E8^E2 regulates its repression activity by a novel mechanism, and this seems to be important for the modulation of host cell gene expression but not viral replication.IMPORTANCE Posttranslational modification of viral proteins is a common feature to modulate their activities. Phosphorylation of serine residues S298 and S301 in the hinge region of the bovine papillomavirus type 1 E2 protein has been shown to restrict viral replication. The papillomavirus E8^E2 protein shares the hinge domain with E2 and acts as a repressor of viral replication. A large fraction of HPV31 E8^E2 is phosphorylated at S78 in the hinge region, and this is important for E8^E2's repression activity. Surprisingly, phosphorylation at S78 in E8^E2 has no impact on viral replication in tissue culture but rather seems to modulate the expression of a small number of cellular genes. This may indicate that phosphorylation of viral transcription factors serves to broaden their target gene specificity.

Control of viral replication and transcription by the papillomavirus E8

Human papillomaviruses have adjusted their replication levels to the differentiation state of the infected keratinocyte. PV genomes replicate in undifferentiated cells at low levels and to high levels in differentiated cells. Genome replication requires the viral E1 helicase and the viral E2 transcription/replication activator. The limited replication in undifferentiated cells is predominantly due to the expression of the highly conserved E8^E2 viral repressor protein, which is a fusion between E8 and the C-terminal half of the E2 protein. E8^E2 is a sequence-specific DNA binding protein that inhibits viral gene expression and viral genome replication. The E8 domain is required for repression activities, which are mainly due to the interaction with cellular NCoR/SMRT corepressor complexes. In the case of HPV16, the most carcinogenic HPV type, E8^E2 not only limits genome replication in undifferentiated cells but also productive replication in differentiated epithelium. E8^E2 is expressed from a separate promoter that is controlled by unknown cellular factors and the viral transcription and replication regulators E1, E2 and E8^E2. In summary, E8^E2 is an important negative regulator whose levels may be critical for the outcome of HPV infections.

Interaction of NCOR/SMRT Repressor Complexes with Papillomavirus E8

Infections with high-risk human papillomaviruses (HR-HPV) such as HPV16 and 31 can lead to ano-genital and oropharyngeal cancers and HPV types from the beta genus have been implicated in the development of non-melanoma skin cancer. HPV replicate as nuclear extrachromosomal plasmids at low copy numbers in undifferentiated cells. HPV16 and 31 mutants have indicated that these viruses express an E8^E2C protein which negatively regulates genome replication. E8^E2C shares the DNA-binding and dimerization domain (E2C) with the essential viral replication activator E2 and the E8 domain replaces the replication/transcription activation domain of E2. The HR-HPV E8 domain is required for inhibiting viral transcription and the replication of the viral origin mediated by viral E1 and E2 proteins. We show now that E8^E2C also limits replication of HPV1, a mu-PV and HPV8, a beta-PV, in normal human keratinocytes. Proteomic analyses identified all NCoR/SMRT corepressor complex components (HDAC3, GPS2, NCoR, SMRT, TBL1 and TBLR1) as co-precipitating host cell proteins for HPV16 and 31 E8^E2C proteins. Co-immunoprecipitation and co-localization experiments revealed that NCoR/SMRT components interact with HPV1, 8, 16 and 31 E8^E2C proteins in an E8-dependent manner. SiRNA knock-down experiments confirm that NCoR/SMRT components are critical for both the inhibition of transcription and HPV origin replication by E8^E2C proteins. Furthermore, a dominant-negative NCoR fragment activates transcription and replication only from HPV16 and 31 wt but not from mutant genomes encoding NCoR/SMRT-binding deficient E8^E2C proteins. In summary, our data suggest that the repressive function of E8^E2C is highly conserved among HPV and that it is mediated by an E8-dependent interaction with NCoR/SMRT complexes. Our data also indicate for the first time that NCoR/SMRT complexes not only are involved in inhibiting cellular and viral transcription but also in controlling the replication of HPV origins.