# A severely affected MRL/Lpr mouse exhibits a divergent clinical-immune profile associated with a profound metabolic alteration

Karim Matmat, Carole Jamey, Daniel Brumaru, Ayikoe-Guy Mensah-Nyagan, Helene Jeltsch-David

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Karim Matmat, Carole Jamey, Daniel Brumaru, Ayikoe-Guy Mensah-Nyagan, Helene Jeltsch-David. A severely affected MRL/Lpr mouse exhibits a divergent clinical-immune profile associated with a profound metabolic alteration. 2026. hal-05450985

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A severely affected MRL/Lpr mouse exhibits a divergent clinical-immune
profile associated with a profound metabolic alteration.

Karim Matmat¹*, Carole Jamey², Daniel Brumaru¹,², Ayikoé-Guy Mensah-Nyagan¹, Hélène Jeltsch-David³*

¹Inserm – Centre d’Investigation Clinique 1434, Neuroprotection and Remyelination – Centre de Recherche en Biomédecine de Strasbourg, University of Strasbourg, Strasbourg, France.

²Laboratoire de Biochimie et de Biologie Moléculaire, Pôle de biologie, Hôpitaux Universitaires de Strasbourg, Strasbourg, France

³Laboratoire des Sciences de l'Ingénieur de l'Informatique et de l'Imagerie ICube, UMR 7357, CNRS / Université de Strasbourg / INSA / ENGEES / INRIA, Strasbourg, France.

*Corresponding Authors: Karim Matmat (matmat@unistra.fr) – ORCID: 0009-0009-8226-4846); Hélène Jeltsch-David (hdavid@unistra.fr – ORCID: 0000-0003-3630-7242)

**Ethics approval statement:** All procedures complied with the European Directive 2010/63/EU and the French governmental decree 2013-118. Experimental protocols were approved by the local Animal Ethics Committee (APAFIS#35144) and conducted under the supervision of authorized investigators in a certified animal facility (Faculty of Medicine, University of Strasbourg).

**Conflict of interest statement:** The authors declare no conflict of interest.

**Funding statement:** Not applicable.

**Data availability statement:** All data supporting the findings of this study are available from the corresponding author upon reasonable request.

**Word count:** 1200
## 23 Abstract

Systemic lupus erythematosus (SLE) displays marked clinical and biological heterogeneity that is incompletely captured by group-based analyses. Although the MRL/Lpr mouse is a well-established lupus model, individual-level divergence within this genetically homogeneous strain remains poorly characterized.

Here, we describe an MRL/Lpr mouse exhibiting an exceptionally severe systemic phenotype and provide an integrative characterization combining clinical assessment, inflammatory profiling, neuroaxonal injury, and targeted metabolomics. Despite pronounced clinical deterioration, including severe proteinuria, reduced organ weights, and marked neuroaxonal damage, this mouse did not show a globally exacerbated cytokine profile relative to other MRL/Lpr animals. In contrast, plasma neurofilament light chain levels were massively elevated, indicating substantial neuroaxonal injury. Targeted metabolomic analysis revealed a profoundly altered biochemical signature, with coordinated disruptions in nitrogen handling, sulfur amino acid metabolism, and neurometabolic pathways, clearly separating this animal from both control and lupus-prone peers.

These findings illustrate that extreme disease severity can emerge independently of overt cytokine escalation and identify metabolic dysregulation as a major dimension of pathological divergence at advanced disease stages. Although descriptive and based on a single individual, this work highlights the value of extreme phenotypes for uncovering biological inflection points that remain concealed in averaged analyses and supports the integration of metabolic readouts alongside inflammatory markers in autoimmune disease research.

## 44 Introduction

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disorder marked by fluctuating inflammatory activity, multisystem involvement, and substantial inter-individual
variability in disease trajectories [1-3]. The MRL/MpJ-Faslpr (MRL/Lpr) mouse strain is one
of the most established spontaneous models of systemic autoimmunity, recapitulating key
features such as lymphoproliferation, glomerulonephritis, neurobehavioral alterations,
dermatitis, and chronic inflammation [4, 5]. Despite its genetic homogeneity this model
nonetheless displays notable variability in disease onset and severity, thereby mirroring
essential aspects of human SLE heterogeneity [6, 7]. Such intra-strain divergence is rarely
explored at the individual level, although unusually severe presentations can reveal mechanisms
undetectable in group-averaged analyses [8]. This is particularly relevant in chronic
autoimmune disease, in which inflammatory burden, metabolic stress, and tissue injury may
diverge substantially between individuals [9].

In this Short Communication, we describe an MRL/Lpr mouse exhibiting an exceptionally
severe systemic phenotype. By integrating clinical, inflammatory, and metabolic assessments,
we illustrate how disease severity can emerge independently of cytokine escalation.

Materials & Methods

Animals and pathological assessment

Female MRL/Lpr and congenic MRL+/+ mice (The Jackson Laboratory, US) were housed
under standard conditions with *ad libitum* access to food and water. At 17-weeks of age,
corresponding to the peak of systemic autoimmunity, animals were anesthetized with isoflurane
and euthanized by decapitation. Blood was collected immediately and plasma was obtained by
centrifugation (2,000 g, 5 min). Kidneys, spleen, brain, and spinal cord were rapidly dissected
and weighed. Terminal proteinuria was assessed using Albustix® reagent strips (Siemens). One
MRL/Lpr mouse (#15) displayed an unusually severe systemic phenotype and was therefore
examined descriptively as an extreme case. All procedures complied with EU Directive
2010/63/EU and were approved by the institutional ethics committee (APAFIS#35144).
71 **Cytokine and NfL quantification**

72 Plasma cytokines levels were quantified using a custom U-PLEX multiplex assay (Meso Scale Discovery, MSD®) and neurofilament light chain (NfL) was measured using a U-PLEX singleplex kit from the same manufacturer. EDTA plasma samples were processed according to the manufacturer's instructions, and electrochemiluminescence signals were acquired on a MESO QuickPlex SQ 120. Concentrations were interpolated from standard curves and expressed in pg/mL.

78 **Targeted metabolomic profiling**

79 Plasma amino acids and related metabolites were quantified using the MassChrom® Amino Acids kit (Chromsystems), a standardized clinical LC-MS/MS method, following the manufacturer's instructions. Final concentrations (µmol/L) were used for downstream analyses.

82 **Statistical analysis**

83 Normality was assessed using the Shapiro-Wilk test and variance homogeneity with Levene's test. Depending on these assumptions, comparisons were performed between MRL+/+ and MRL/Lpr mice using an unpaired t-test, Welch's t-test, or a Mann-Whitney test. PCA was computed on scaled cytokine or metabolite data. The composite inflammatory score was generated by z-normalizing each cytokine. Mouse #15 was included in the MRL/Lpr group for all statistical analyses; its profile was then described qualitatively to illustrate intra-group variability. Analyses were performed in R (v4.3).

90 **Results**

91 **Clinical phenotype reveals a severely affected MRL/Lpr mouse.**

92 At terminal assessment, MRL/Lpr mice showed the expected systemic alterations relative to MRL+/+ controls. Body weight was broadly comparable within the MRL/Lpr group, although
94 mouse #15 exhibited the lowest weight of all animals (Figure 1a). Kidney, spleen, and spinal
95 cord weights were significantly increased in MRL/Lpr mice, along with elevated proteinuria
96 (Figure 1b-c). Interestingly, mouse #15 displayed an atypical organ pattern: kidney weight
97 remained within the typical MRL/Lpr range, but spleen, brain, and spinal cord weights were
98 the lowest of the group, together with a maximal proteinuria score (5/5). This animal also
99 showed severe ulcerative dermatitis with complete loss of snout fur, associated with a visibly
100 deteriorated general condition (Figure 1d).

101 *Inflammatory profiling shows limited cytokine dysregulation in mouse #15, but*
102 pronounced neuro-axonal injury.

103 Principal component analysis (PCA) of plasma cytokines did not separate mouse #15 from the
104 remainder of the MRL/Lpr group (Figure 1e), indicating that its marked clinical deterioration
105 was not associated with an overtly exaggerated cytokine profile. Other animals, particularly
106 #20 and #22, exhibited more divergent inflammatory signatures. Nevertheless, the radar plot
107 revealed selective increases in TNFα and IL-1β in mouse #15, with values exceeding the
108 MRL/Lpr group mean and ranking among the highest in the cohort, notably, TNFα reached the
109 maximum recorded level (Figure 1f-h). Consistently, the composite inflammatory score
110 confirmed this pattern, placing mouse #15 in the upper range of group variability without
111 exceeding the expected dispersion (Figure 1i).

112 In contrast, plasma NfL levels showed a striking divergence: mouse #15 displayed a massive
113 elevation, far surpassing all other animals (Figure 1j). This divergence indicates that neuro-
114 axonal injury can escalate independently of systemic cytokine levels.

115 *Metabolic profiling reveals a profoundly altered signature in mouse #15.*

116 Targeted metabolomics showed a strongly altered biochemical profile in mouse #15. PCA
117 clearly isolated this animal from both MRL+/+ controls and the remaining MRL/Lpr mice,
indicating a global shift in amino-acid homeostasis (Figure 2a). This separation reflects a coordinated shift across multiple metabolites rather than isolated abnormalities. Fold-change analysis (e.g., |log₂FC| > 2) identified major elevations in citrulline, alanine, and 3-methylhistidine, reflecting disruptions in nitrogen handling and muscle-derived metabolites, together with marked increases in cystathionine and taurine, indicative of altered sulfur amino-acid metabolism (Figure 2b-c). Conversely, several metabolites were markedly decreased, including a near-complete depletion of GABA. Pathway enrichment of metabolites meeting the |log₂FC| > 2 threshold highlighted significant over-representation of the urea cycle, glutamate metabolism, ammonia recycling, and related nitrogen-processing pathways (Figure 2d).

Together, these alterations delineate a profoundly dysregulated metabolic phenotype. This pattern suggests that severe metabolic dysregulation may better capture late-stage deterioration than inflammatory cytokine measures. An integrative schematic summarizing the constellation of clinical, inflammatory, and metabolic abnormalities observed in mouse #15 is provided in Figure 2e.

## Discussion

This study illustrates that, even within a highly penetrant autoimmune model, an extreme systemic phenotype can emerge without a corresponding escalation in inflammatory activity. Although based on a single individual, documenting such an extreme presentation remains informative, highlighting biological divergence within a genetically homogeneous strain. These findings suggest that, at advanced disease stages, systemic inflammatory markers may reach a plateau beyond which they no longer discriminate severity, thereby limiting their capacity to capture divergent pathological trajectories [9, 10].

In contrast, metabolic profiling revealed a distinctly altered biochemical landscape that clearly set this mouse apart from all other. The magnitude and coherence of these metabolic
abnormalities, including coordinated disruptions in urea-cycle-related nitrogen handling, sulfur amino-acid metabolism, and muscle-derived metabolites, indicate that downstream metabolic dysfunction may serve as a more discriminating correlate of extreme systemic deterioration. This aligns with observations in human lupus, where metabolic phenotypes often diverge independently of inflammatory indices [11–13], supporting the notion that metabolic dysregulation becomes a predominant dimension of disease heterogeneity associated with extreme disease severity once inflammation stabilizes at chronically elevated levels. In this context, metabolic readouts may offer greater sensitivity for detecting late-stage systemic deterioration than cytokine measurements alone.

Overall, this work underlines the importance of integrating metabolic assessments alongside inflammatory markers in autoimmune research. Extreme phenotypes can reveal mechanistic inflection points that remain concealed in group-averaged analyses, thereby enhancing the translational relevance of preclinical models. Although causality cannot be inferred from a single observation, the coherence of the clinical and metabolic alterations strengthens its value as a representative extreme phenotype. Such rare divergences highlight the importance of considering individual-level variation when interpreting autoimmune phenotypes.

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162 Author Contributions

KM: Conceptualization, data curation, formal analysis, investigation, visualization, writing – original draft, writing – review & editing. CJ: Investigation, writing – review & editing. DB:
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Investigation, writing – review & editing. AGMN: Investigation, writing – review & editing.

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HJD: Conceptualization, investigation, writing –review & editing.

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## Figure legends

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**Figure 1.** Systemic and behavioral profile of MRL/lpr mice at 17 weeks, illustrating the markedly aggravated condition of mouse #15. (a–c) Body and organs weights, and terminal proteinuria in MRL+/+ and MRL/lpr mice. Brain and spinal cord weights are included to document central nervous system tissue mass at endpoint. (d) Representative photographs showing the external phenotype of an MRL+/+ mouse, a typical MRL/lpr mouse, and mouse #15 at sacrifice. (e) Principal component analysis (PCA) integrating all measured inflammatory cytokines. (f) Radar plot summarizing cytokine levels across groups, with the profile of mouse #15 highlighted. (g–h) Circulating concentrations of TNFα (g) and IL-1β (h). (i) Composite inflammatory score derived from normalized cytokine values. (j) Plasma neurofilament light chain (NfL) levels as a marker of neuroaxonal injury. Data are presented as individual values with mean ± SEM. Statistical procedures are detailed in the Methods section. Across all scatterplots, mouse #15 is represented as a triangle and all other individuals as circles. Data are presented as individual values with mean ± SEM. Statistical procedures are detailed in the Methods section.

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**Figure 2.** Metabolic profiling reveals a distinct biochemical signature in mouse #15. (a) PCA of plasma metabolites in MRL+/+ and MRL/lpr mice, highlighting the isolated metabolic position of mouse #15. (b) Log₂ fold-change distribution of all quantified metabolites comparing mouse #15 with the remaining MRL/lpr mice, illustrating the amplitude and direction of metabolic deviations. (c) Representative individual metabolite concentrations, including markers of nitrogen handling (urea cycle), sulfur amino-acid metabolism, muscle catabolism, and neurochemical imbalance. Mouse #15 is displayed as a triangle across all

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189 scatterplots. (d) Pathway enrichment analysis based on altered metabolites, highlighting over-represented metabolic pathways (FDR-adjusted). (e) Integrative schematic summarizing the systemic phenotype of mouse #15, linking major clinical features with the corresponding metabolic alterations. Data are shown as individual values with mean ± SEM. Statistical procedures are described in the Methods.

194 References

195 [1] Sammaritano LR, Askanase A, Bermas BL, et al. 2025 American College of Rheumatology (ACR) Guideline for the Treatment of Systemic Lupus Erythematosus. *Arthritis Care & Research* 2025; acr.25690.

198 [2] Tsokos GC. Systemic Lupus Erythematosus. *N Engl J Med* 2011; 365; 2110-21.

199 [3] Dai X, Fan Y, Zhao X. Systemic lupus erythematosus: updated insights on the pathogenesis, diagnosis, prevention and therapeutics. *Sig Transduct Target Ther* 2025; 10; 102.

202 [4] Jeltsch-David H, Muller S. Neuropsychiatric systemic lupus erythematosus and cognitive dysfunction: The MRL-lpr mouse strain as a model. *Autoimmunity Reviews* 2014; 13; 963-73.

204

205 [5] Perry D, Sang A, Yin Y, et al. Murine models of systemic lupus erythematosus. *J Biomed Biotechnol* 2011; 2011; 271694.

207 [6] Cabana-Puig X, Bond JM, Wang Z, et al. Phenotypic Drift in Lupus-Prone MRL/lpr Mice: Potential Roles of MicroRNAs and Gut Microbiota. *Immunohorizons* 2022; 6; 36-46.

209 [7] Richard ML, Gilkeson G. Mouse models of lupus: what they tell us and what they don't. *Lupus Sci Med* 2018; 5; e000199.

211 [8] Banchereau R, Hong S, Cantarel B, et al. Personalized Immunomonitoring Uncovers Molecular Networks that Stratify Lupus Patients. *Cell* 2016; 165; 1548-50.
213

[9] Wang FQ, Dang X, Yang W. Transcriptomic studies unravel the molecular and cellular complexity of systemic lupus erythematosus: A review. *Clinical Immunology* 2024; 268; 110367.

214

215

216

[10] Crow MK. Type I interferon in the pathogenesis of lupus. *J Immunol* 2014; 192; 5459-68.

217

[11] Zhang W, Zhao H, Du P, et al. Integration of metabolomics and lipidomics reveals serum biomarkers for systemic lupus erythematosus with different organs involvement. *Clinical Immunology* 2022; 241; 109057.

218

219

220

[12] Terrell M, Morel L. The Intersection of Cellular and Systemic Metabolism: Metabolic Syndrome in Systemic Lupus Erythematosus. *Endocrinology* 2022; 163; bqac067.

221

222

[13] Zhang C, Wang H, Yin L, et al. Immunometabolism in the pathogenesis of systemic lupus erythematosus. *Journal of Translational Autoimmunity* 2020; 3; 100046.

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