Presenilin1 regulates Th1 and Th17 effector responses but is not required for experimental autoimmune encephalomyelitis

Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) where pathology is thought to be regulated by autoreactive T cells of the Th1 and Th17 phenotype. In this study we sought to understand the functions of Presenilin 1 (PSEN1) in regulating T cell effector responses in the experimental autoimmune encephalomyelitis (EAE) murine model of MS. PSEN1 is the catalytic subunit of γ-secretase a multimolecular protease that mediates intramembranous proteolysis. γ-secretase is known to regulate several pathways of immune importance. Here we examine the effects of disrupting PSEN1 functions on EAE and T effector differentiation using small molecule inhibitors of γ-secretase (GSI) and T cell-specific conditional knockout mice (PSEN1 cKO). Surprisingly, blocking PSEN1 function by GSI treatment or PSEN1 cKO had little effect on the development or course of MOG35-55-induced EAE. In vivo GSI administration reduced the number of myelin antigen-specific T cells and suppressed Th1 and Th17 differentiation following immunization. In vitro, GSI treatment inhibited Th1 differentiation in neutral but not IL-12 polarizing conditions. Th17 differentiation was also suppressed by the presence of GSI in all conditions and GSI-treated Th17 T cells failed to induce EAE following adoptive transfer. PSEN cKO T cells showed reduced Th1 and Th17 differentiation. We conclude that γ-secretase and PSEN1-dependent signals are involved in T effector responses in vivo and potently regulate T effector differentiation in vitro, however, they are dispensable for EAE.


INTRODUCTION
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.

METHODS
Ethical statement 5 Indicate the nature of the ethical review permissions, relevant licences (e.g. Animal [Scientific Procedures] Act 1986), and national or institutional guidelines for the care and use of animals, that cover the research.

Study design 6
For each experiment, give brief details of the study design including: a. The number of experimental and control groups. b. Any steps taken to minimise the effects of subjective bias when allocating animals to treatment (e.g. randomisation procedure) and when assessing results (e.g. if done, describe who was blinded and when). c. The experimental unit (e.g. a single animal, group or cage of animals). A time-line diagram or flow chart can be useful to illustrate how complex study designs were carried out.

7
For each experiment and each experimental group, including controls, provide precise details of all procedures carried out. For example: a. How (e.g. drug formulation and dose, site and route of administration, anaesthesia and analgesia used [including monitoring], surgical procedure, method of euthanasia). Provide details of any specialist equipment used, including supplier(s). b. When (e.g. time of day). c. Where (e.g. home cage, laboratory, water maze). d. Why (e.g. rationale for choice of specific anaesthetic, route of administration, drug dose used).
Experimental animals 8 a. Provide details of the animals used, including species, strain, sex, developmental stage (e.g. mean or median age plus age range) and weight (e.g. mean or median weight plus weight range). b. Provide further relevant information such as the source of animals, international strain nomenclature, genetic modification status (e.g. knock-out or transgenic), genotype, health/immune status, drug or test naïve, previous procedures, etc. temperature, quality of water etc for fish, type of food, access to food and water, environmental enrichment). 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. b. Describe the order in which the animals in the different experimental groups were treated and assessed.

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.

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 ).
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. 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.