Downregulation of TNIP1 Expression Leads to Increased Proliferation of Human Keratinocytes and Severer Psoriasis-Like Conditions in an Imiquimod-Induced Mouse Model of Dermatitis

Psoriasis is a chronic, inflammatory skin disease involving both environmental and genetic factors. According to genome-wide association studies (GWAS), the TNIP1 gene, which encodes the TNF-α–induced protein 3-interacting protein 1 (TNIP1), is strongly linked to the susceptibility of psoriasis. TNIP1 is a widely expressed ubiquitin sensor that binds to the ubiquitin-editing protein A20 and restricts TNF- and TLR-induced signals. In our study, TNIP1 expression decreased in specimens of epidermis affected by psoriasis. Based on previous studies suggesting a role for TNIP1 in modulating cancer cell growth, we investigated its role in keratinocyte proliferation, which is clearly abnormal in psoriasis. To mimic the downregulation or upregulation of TNIP1 in HaCaT cells and primary human keratinocytes (PHKs), we used a TNIP1 specific small interfering hairpin RNA (TNIP1 shRNA) lentiviral vector or a recombinant TNIP1 (rTNIP1) lentiviral vector, respectively. Blocking TNIP1 expression increased keratinocyte proliferation, while overexpression of TNIP1 decreased keratinocyte proliferation. Furthermore, we showed that TNIP1 signaling might involve extracellular signal-regulated kinase1/2 (Erk1/2) and CCAAT/enhancer-binding protein β (C/EBPβ) activity. Intradermal injection of TNIP1 shRNA in BALB/c mice led to exaggerated psoriatic conditions in imiquimod (IMQ)-induced psoriasis-like dermatitis. These findings indicate that TNIP1 has a protective role in psoriasis and therefore could be a promising therapeutic target.


BACKGROUND AND PURPOSE: Psoriasis is a chronic, inflammatory skin disease involving both
TNFa-induced protein 3-interacting protein 1 exaggerates imiquimod-induced psoriasis-like dermatitis in mice when downregulated environmental and genetic factors. GWAS revealed that TNIP1 gene is strongly linked to the susceptibility of psoriasis. We revealed that TNIP1 expression is downregulated in psoriasis skin when compared with normals. Furthermore, we discovered that downregulated TNIP1 increased keratinocyte proliferation while overexpression of TNIP1 decreased keratinocyte proliferation. In the animal experiment, our aim was to evaluate the downregulated TNIP1 whether protected or exaggerated imiquimod (IMQ) induced psoriatic dermatitis in mice. EXPERIMENTAL APPROACH: Mice at 8 to 10 weeks of age received intradermal injections of RFP-tagged lentiviral particles encoding shRNA to TNIP1 (7.5×107TU) or control shRNA (7.5×107TU) on their back skin. 7 d later, starting the topical application of IMQ, for 6 consecutive days, then the mice sacrificed and skin sample around injection area were collected for HE staining. We recorded the clinical manifestation of mice during the treatment of IMQ and have skin sections for pathological examination.

Background
3 a. Include sufficient scientific background (including relevant references to previous work) to understand the motivation and context for the study, and explain the experimental approach and rationale.
b. Explain how and why the animal species and model being used can address the scientific objectives and, where appropriate, the study's relevance to human biology.

Objectives 4
Clearly describe the primary and any secondary objectives of the study, or specific hypotheses being tested.
KEY RESULTS: We have observed that mice injected TNIP1 shRNA showed much more severe inflammation during the consecutive treatment of IMQ, and histological examination of skin sections from these mice showed much more significant epidermal hyperplasia.
CONCLUSIONS AND IMPLICATIONS: These results highlighted that TNIP1 exaggerated IMQ-induced psoriasis-like dermatitis in mice when downregulated and suggest TNIP1 may be a potential therapeutic target for psoriasis.
Psoriasis is a common chronic inflammatory skin disorder affecting 1-2% of the northern American and European populations [1], and the TNIP1 gene, which encodes protein TNIP1, has been revealed to be susceptibility gene of psoriasis [2,3]. However the mechanisms of how the susceptibility gene and the encoded protein contribute to the pathogenesis of psoriasis remain largely unclear. Psoriasis is characterized by hyperproliferation and aberrant terminal differentiation of keratinocytes, and the role of TNIP1 in proliferation of keratinocyte has never been explored. We revealed that TNIP1 expression is downregulated in psoriasis skin when compared with normals, and discovered that downregulated TNIP1 increased keratinocyte proliferation while overexpression of TNIP1 decreased keratinocyte proliferation. Furthermore we intended to explore whether TNIP1 involved in the pathogenesis of psoriasis especially by targeting keratinocytes in vivo.
For this purpose, we selected the IMQ-induced psoriatic dermatitis mice model [4,5], which has been widely accepted to study erythema, scales and psoriatic plaque formation.
Therefore, the objective of this study was to determine whether TNIP1 involved in pathogenesis of psoriasis and whether the downregulated TNIP1 protected or exaggerated IMQ induced psoriatic dermatitis in mice. All animal studies were approved by the animal ethics committee of the Third Military Medical University according to Dutch legislation on animal experiments. Mice were anaesthetized with sodium pentobarbital when necessary, and all efforts were made to minimize suffering.

METHODS
There're three groups, 3 in each group.
Animals were randomized into treatment groups by picking numbers out of a hat.
In the study, n refers to number of animals. The lentiviral particles (7.5×107TU) were intradermally injected into mice skin via microliter inyeetor(50ul). IMQ cream (5%) was commercial products (Mingxin, Sichuan, China) and was topically applied on back skin of mice, once a day for 6 consecutive days. Mice were anaesthetized with sodium pentobarbital (50mg/kg, i.p.) when necessary.
Treatment were conducted at about 10:00 A.M. in the morning.
Experiment were conducted under Laminar Flow Hood in the SPF animal house where the mice were maintained.
To observe the clinical manifestation of mice at scheduled timepoint.
BALB/c mice were purchased from the laboratory animal center of the Third Military Medical University (Chongqing, China) and Vendor health reports indicated that the mice were free of known viral, bacterial and parasitic pathogens. c. Welfare-related assessments and interventions that were carried out prior to, during, or after the experiment.
Sample size 10 a. Specify the total number of animals used in each experiment, and the number of animals in each experimental group.
b. Explain how the number of animals was arrived at. Provide details of any sample size calculation used.
c. Indicate the number of independent replications of each experiment, if relevant Allocating animals to experimental groups 11 a. Give full details of how animals were allocated to experimental groups, including randomisation or matching if done.
Mice were housed with an inverse 12 hours day-night cycle with lights on at 8:30pm in a temperature (22±1oC) and humidity (55±5%) controlled room.
All mice were allowed free access to water and a maintenance diet containing 0.75% calcium in a 12-hour light/dark cycle, with room temperature at 21±2 °C. All cages contained wood shavings, bedding.
All cages contained a cardboard tube for environmental enrichment.
Nine male healthy mice were divided into three groups of three each. Animals of group I received TNIP1 shRNA lentivirus injection and lotion control topical application, group II animals received control shRNA lentivirus injection and IMQ topical application, and group III received TNIP1 shRNA lentivirus injection and IMQ topical application for six days.
We performed the experiments as before mentioned for we only have 9 mice met the age standard.
significant, which were sufficient to draw our conclusion that downregulated TNIP1 IMQ-induced psoriatic dermatitis in mice. So we did not repeat the experiment and n=3.
The experiment was not repeated due to unnecessary.
The animals were allocated to experimental groups randomly.
We decided the sample size by two groups of completely random design of sample mean comparison.
the clinical and pathological menifestation showed the same trend and the difference were Since exaggerated temperature (21± 2°C) and humidity (55±5%) controlled room.
b. Describe the order in which the animals in the different experimental groups were treated and assessed.

Experimental outcomes
12 Clearly define the primary and secondary experimental outcomes assessed (e.g. cell death, molecular markers, behavioural changes).
Statistical methods 13 a. Provide details of the statistical methods used for each analysis.
b. Specify the unit of analysis for each dataset (e.g. single animal, group of animals, single neuron).
c. Describe any methods used to assess whether the data met the assumptions of the statistical approach.

RESULTS
Baseline data 14 For each experimental group, report relevant characteristics and health status of animals (e.g. weight, microbiological status, and drug or test naïve) prior to treatment or testing. (This information can often be tabulated).
Numbers analysed 15 a. Report the number of animals in each group included in each analysis. Report absolute numbers (e.g. 10/20, not 50% 2 ).
The order: intradermal injection of lentiviral particles---7 d later, starting the topical application of IMQ, for 6 consecutive days-mice sacrificed for HE staining.
One primary outcome measures was analyzed: Clinical scores of IMQ-induced psoriatic dermatitis. In addition, three secondary outcome measures were evaluated: epidermis thickness, neutrophil infiltration and parakeratosis.
Data were analyzed using SPSS 13.0 software, and results are expressed as mean±standard deviation (SD). Statistical differences between the groups were assessed by one-way analysis of variance (ANOVA) followed by Duncan's Multiple Range test. Differences were considered significant when P<0.05 or P<0.01.
For each test, the experimental unit was an individual animal.
BALB/c mice were purchased from the laboratory animal center of the Third Military Medical University (Chongqing, China) and were maintained under specific pathogen-free conditions according to standard laboratory procedures. The mice were free of all viral, bacterial,and parasitic pathogens, except for Helicobacter species.
There are three groups, and n=3 in each group.
In the first round of animal experiment, we randomly allocated three mice in each group. During the consecutive 6 days IMQ treatment, the clinical manifestation of mice (erythema, scales, and thickness of skin) showed significant differences between the three groups since the third day. We did not repeat the experiment later because it's already sufficient to draw the conclusion that downregulated TNIP1 exaggerated IMQ-induced psoriatic dermatitis in mice, or the mice would sacrifice unnecessarily.
b. If any animals or data were not included in the analysis, explain why.

Outcomes and estimation
16 Report the results for each analysis carried out, with a measure of precision (e.g. standard error or confidence interval).
Adverse events 17 a. Give details of all important adverse events in each experimental group.
b. Describe any modifications to the experimental protocols made to reduce adverse events. - No animals or data were not included.
No obvious adverse events were observed in every experimental group.
To prepare the same volume and same amount of lentiviral particles (TNIP1 shRNA and Con shRNA) before injection will make the protocol more standardized. And higher titer means less volume, which will reduce the skin burden from intradermal injection. c. Describe any implications of your experimental methods or findings for the replacement, refinement or reduction (the 3Rs) of the use of animals in research.

Generalisability/ translation
19 Comment on whether, and how, the findings of this study are likely to translate to other species or systems, including any relevance to human biology.
Funding 20 List all funding sources (including grant number) and the role of the funder(s) in the study.
The aim of our study was to verify whether TNIP1 could exaggerate or suppress the imiquimod-induced psoriasis-like dermatitis in mice when downregulated. Our study revealed that mice with decreased TNIP1 were more vulnerable to IMQ-induced psoriasis. These findings are consistent with Callahan's study, in which the authors distinctively knockout TNIP1in dendritic cells.
A limitation of this study is the fact that lentivirus infection is quite unspecific in vivo: it not only infects keratinocyte, but also other epidermal cells, including dendritic cells. So the exaggerated IMQ-induced psoriatic dermatitis cannot be only interpreted as a keratinocyte phenomenon.
If we could knockout TNIP1 in distinctive cell type, for instance, keratinocyte, the results would be more supportive and convinced. Thus the sacrifice of mice would be more valuable.
We delivered the lentiviral particles by intradermal injection, and discovered that lenvirus infected not only mice skin but also kidney and spleen. This offered the possibility to study the effects of TNIP1 in systemic lupus erythematosus (SLE), in which kidney was clearly abnormal.