🧠 neuroscience

Overnight Th1 immune activation predicts the cortisol awakening response

This pilot study involving three healthy subjects provides exploratory evidence that overnight Th1 immune activation, measured by neopterin levels, positively predicts the magnitude of the cortisol awakening response, supporting the immunological awakening hypothesis.

Original authors: Seizer, L., Matuskov, M. G., Gostner, J., Schubert, C.

Published 2026-07-09

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CC BY 4.0

Original authors: Seizer, L., Matuskov, M. G., Gostner, J., Schubert, C.

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ⚕️ This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer

The Big Idea: Your Body's Morning Alarm Clock and Its "Night Watch"

Imagine your body has two main systems that need to switch gears every morning:

The Energy System (Cortisol): This is your internal alarm clock. When you wake up, your body releases a hormone called cortisol to give you a burst of energy, wake up your brain, and get you ready for the day. This sudden jump is called the Cortisol Awakening Response (CAR).
The Defense System (Immune System): While you sleep, your immune system is like a night watch. It gets busy patrolling, looking for invaders, and repairing damage. A specific marker for this "night shift" activity is a substance called neopterin.

The Question:
Scientists have always wondered: Does the work your immune system does while you sleep affect how hard your body hits the "snooze button" and then jumps out of bed in the morning?

The Experiment: A Month of Self-Tracking

This study was a small "pilot" test (like a practice run before a big game) involving just three healthy people. To get very accurate data, the researchers didn't just ask them how they felt; they had them track their bodies for 30 days in their own homes.

Here is what the participants did every single night and morning:

The Night Shift: They collected their urine overnight to measure neopterin (the "night watch" activity).
The Morning Rush: They wore a smart headband (like a sleep tracker) to know the exact second they woke up. Then, they spit into a cup three times: right when they woke up, 30 minutes later, and 45 minutes later. This measured their cortisol levels.

The Discovery: A Stronger Night Watch Means a Louder Alarm

The researchers found a clear connection: When the immune system was more active during the night (higher neopterin), the morning cortisol "jump" was stronger.

Think of it like this:

If your night watch (immune system) had a quiet, easy night, your morning alarm (cortisol) might be a gentle "good morning."
But if your night watch was very busy fighting off potential threats or doing heavy repairs, your body responds by hitting the morning alarm much harder. It's as if your body says, "Wow, we had a lot of work to do last night; we need a massive energy boost to get back to normal daytime mode!"

Why This Matters (According to the Paper)

The paper suggests this isn't just a random glitch. It supports a theory called the "Immunological Awakening Hypothesis."

Imagine your body is a city. At night, the city's repair crews (immune system) are out working. In the morning, the mayor (cortisol) needs to come out and give a big speech to tell everyone to stop repairing and start working their day jobs. The study suggests that if the repair crews worked extra hard, the mayor has to give an even louder, more energetic speech to get the city back on track.

Important Caveats (The "Fine Print")

The authors are very careful to say two things:

It's a Pilot Study: This was only done on three people. It's like testing a new recipe on three friends before serving it to a whole restaurant. The results look promising, but we need to test it on hundreds of people to be sure.
It's Exploratory: The paper doesn't claim this explains diseases or how to treat patients yet. It simply provides the first piece of evidence that the immune system and the morning stress hormone are talking to each other.

In short: This study is the first to show that how hard your immune system works while you sleep might determine how big of a "jump" your energy levels take when you wake up.

Technical Summary: Overnight Th1 Immune Activation Predicts the Cortisol Awakening Response

Problem and Background
The Cortisol Awakening Response (CAR) is characterized by a sharp increase in cortisol levels within 30–45 minutes of waking, marking the transition from rest to the active phase. While its exact function remains debated, hypotheses suggest roles in cognitive mobilization and an "immunological awakening" to regulate circadian immune rhythms. Previous research has established that immune activity follows a circadian pattern, with Th1-type pro-inflammatory markers (e.g., IFN-γ, neopterin, TNF-α) peaking during the night, while anti-inflammatory glucocorticoids peak during the day. Although bidirectional communication between the immune system and the Hypothalamic-Pituitary-Adrenal (HPA) axis is known, existing studies investigating the relationship between CAR and immune markers have been limited by their temporal design. Most prior work measured cytokines simultaneously with or after cortisol sampling, or on different days, failing to isolate whether overnight immune activity acts as a modulator of the subsequent morning's CAR. Furthermore, CAR expression is highly sensitive to state variables (62–82% of variance), yet many studies rely on between-subject designs that may obscure these dynamic, within-person fluctuations.

Methodology
This pilot study employed an intensive longitudinal design (integrative single-case study) to investigate within-subject associations between overnight immune activation and the following morning's CAR.

Participants: Three healthy adults (2 female, 1 male; mid-twenties).
Duration: 30 days of data collection per participant in everyday life settings.
Immune Marker: Overnight urinary neopterin was used as a biomarker for Th1 immune activation (specifically IFN-γ stimulated macrophage/dendritic cell activity). Participants collected first-morning urine after discarding pre-sleep voids. Neopterin excretion rates were calculated via high-performance liquid chromatography (HPLC).
CAR Measurement: Saliva was collected via passive drool at 0, 30, and 45 minutes post-awakening. Cortisol levels were quantified using ELISA. The dynamic increase was calculated using two metrics: Area Under the Curve with respect to increase (AUCI) and Mean Increase (MnInc).
Objective Awakening: Participants wore a Dreem 3 EEG headband overnight to objectively determine the exact awakening time. Data points where the delay between objective awakening and first saliva sampling exceeded 5 minutes were excluded.
Statistical Analysis: Random-effects regression models were computed. To isolate state effects, overnight neopterin levels were person-mean centered. Models tested the effect of overnight neopterin on CAR (AUCI and MnInc) and first-sample cortisol. Autoregressive controls (previous day's CAR) and idiographic (single-subject) analyses were also performed.

Key Results

Data Quality: Compliance was high (94.67% for saliva, 93.33% for urine). After excluding 12 days due to sampling delays, the final dataset comprised 221 cortisol samples, 84 urine samples, and 75 valid days across all participants.
Primary Finding: There was a significant positive effect of overnight neopterin levels on the CAR.
- For CAR as AUCI: $b = 0.15$ , 95% CI [0.01, 0.29], $p = .032$ .
- For CAR as MnInc: $b = 0.01$ , 95% CI [0.00, 0.01], $p = .041$ .
- These results indicate that higher-than-usual overnight neopterin levels (Th1 activation) predict a stronger dynamic increase in cortisol the following morning.
Robustness: The significance and direction of the effect remained unchanged when controlling for the previous day's CAR. Idiographic models for each individual confirmed that while effect magnitudes varied, the positive direction was consistent across all three participants.

Significance and Claims
The authors claim these findings provide the first evidence supporting the "immunological awakening hypothesis," suggesting that the CAR may serve a physiological role in transitioning the immune system from a nocturnal pro-inflammatory state to a daytime anti-inflammatory setpoint. Specifically, the data implies that higher overnight immune activation necessitates a stronger glucocorticoid signal upon waking to facilitate this recalibration.

The study highlights the utility of intensive longitudinal designs in psychoneuroimmunology to disentangle state-dependent immune-endocrine interactions from stable trait differences. The authors posit that these results have implications for interpreting the CAR as a biomarker in clinical research; altered CAR patterns in psychiatric or inflammatory disorders may reflect disrupted endocrine-immune coupling rather than isolated HPA-axis dysfunction.

Limitations and Modesty
The paper explicitly frames these findings as exploratory and preliminary due to the small sample size ( $N=3$ ). The authors caution that the results cannot be generalized to the wider population without replication in larger cohorts. Additionally, they note that urinary neopterin captures only IFN-γ-associated cellular activation and provides an integrated overnight signal, lacking the temporal resolution to pinpoint specific timing of immune changes throughout the night. Unmeasured time-varying confounders (e.g., sleep quality, stress, menstrual cycle) may also influence the observed associations.