Interferon‐gamma contributes to disease progression in the Ndufs4(−/−) model of Leigh syndrome

Leigh syndrome (LS), the most common paediatric presentation of genetic mitochondrial dysfunction, is a multi‐system disorder characterised by severe neurologic and metabolic abnormalities. Symmetric, bilateral, progressive necrotizing lesions in the brainstem are defining features of the disease. Patients are often symptom free in early life but typically develop symptoms by about 2 years of age. The mechanisms underlying disease onset and progression in LS remain obscure. Recent studies have shown that the immune system causally drives disease in the Ndufs4(−/−) mouse model of LS: treatment of Ndufs4(−/−) mice with the macrophage‐depleting Csf1r inhibitor pexidartinib prevents disease. While the precise mechanisms leading to immune activation and immune factors involved in disease progression have not yet been determined, interferon‐gamma (IFNγ) and interferon gamma‐induced protein 10 (IP10) were found to be significantly elevated in Ndufs4(−/−) brainstem, implicating these factors in disease. Here, we aimed to explore the role of IFNγ and IP10 in LS.

Patients are typically born healthy but develop symptoms of neurodegeneration early in childhood, with symptoms often first appearing after a viral infection or fever [7].Clinical symptoms of LS can include seizures and lactic acidosis, as well as respiratory dysfunction, which results from brainstem disease and is often the ultimate cause of death [1,3].
LS is a genetically diverse disease.Over 110 unique genetic lesions distributed among the nuclear and mitochondrial genomes have been causally linked to LS [8].Homozygosity for loss of function mutations in NDUFS4, which encodes a structural/assembly component of mitochondrial electron transport chain complex I (ETC CI), is one known cause of LS in humans [9].Homozygous loss of Ndufs4 in mice results in a phenotype remarkably consistent with human LS [10].As in human patients, Ndufs4(À/À) mice are born healthy and develop neurologic symptoms early in life.In the Ndufs4(À/À) mouse model, LS-like symptoms arise beginning around 5-6 weeks of age.
Pharmacologic depletion of circulating and tissue macrophages (including cerebral microglia) in Ndufs4(À/À) mice with high doses of the Csf1r (Colony stimulating factor 1 receptor) inhibitor pexidartinib prevents the formation of necrotic lesions, prevents neurologic disease symptoms, and dramatically prolongs lifespan [13].In pexidartinib treated animals, drug toxicity, rather than LS-like disease, is lifespan limiting.Csf1r is critical for the survival and function of macrophages, including brain resident microglia.In recent work, we have established that peripheral macrophages and brain resident microglia both contribute to CNS lesions and drive disease in the Ndufs4(À/À) model [14,15].Cytokine profiling in these studies revealed that interferongamma (IFNγ) and IFNγ-induced protein 10 (IP10, also known as Cxcl10) are significantly elevated in Ndufs4(À/À) mice with overt disease compared to age-matched control animals [13].IFNγ is produced in response to viral infection, signals through the IFNγ receptor (IFNγR), and has pleiotropic functions in the innate and adaptive immune systems including upregulation of interferon-stimulated genes such as IP10/CXCL10 [16].IP10 acts through the chemokine receptor CXCR3 and, among other functions, is known to act as a potent chemoattractant molecule for leukocytes including monocytes/macrophages, T cells and NK cells [17,18].
Here, we assess the roles of IFNγ and IP10 in the pathogenesis of LS via genetic disruption of Ifng and IP10 using knockout lines crossed into the Ndufs4(À/À) model.

Loss of IP10 does not attenuate disease in the Ndufs4 (À/À) mouse model of LS
To evaluate the role of IP10 in the pathogenesis of LS, we generated and assessed disease onset and progression in Ndufs4(À/À) animals with wildtype, heterozygous knockout or homozygous knockout of IP10.This was accomplished by generating a double mutant IP10 knockout Ndufs4 knockout line (IP10 knockout: Jackson laboratory strain 002287; see Methods).qPCR analysis of IP10 expression in brain tissue revealed no difference in IP10 transcript levels in Ndufs4 (À/À)/IP10(+/+) and Ndufs4(À/À)/IP10(+/À) mice.IP10 expression was not detected in Ndufs4(À/À) mice homozygous for deletion of IP10, confirming the effect of the knockout allele (Figure 1A).
• IFNγ contributes to mitochondrial disease and may represent a potential therapeutic target in some settings.
Reduced body weight has previously been reported as a consequence of Ifng deficiency [19].Neurological symptom onset (ataxia and forelimb clasping) was modestly attenuated by homozygous loss of Ifng (Figure 3E,F; see Methods), and rotarod performance was marginally improved by Ifng knockout at P50, though benefits were not maintained to later ages of P60 and 70 (Figure 3H).

DISCUSSION
Here, we used genetic models to assess the role of IP10 and IFNγ in the onset and progression of disease in the Ndufs4(À/À) model of LS.Our data indicate that the leukocyte chemoattractant IP10 is dispensable for both disease onset and progression, while IFNγ, an upstream regulator of IP10 and an important factor in both innate and adaptive immunity, contributes modestly but significantly to weight loss, survival and onset/progression of neurologic symptoms.
Brainstem lesions in Ndufs4(À/À)/Ifng(À/À) may be subtly different by IHC.In the animals assessed no significant differences in lesion area were observed, but the lesions in the Ifng(À/À) animals do appear to have modestly reduced overall lesion cellularity and larger Iba1(+) cells.These changes are somewhat reminiscent of histological changes to brainstem lesions observed in Ndufs4(À/À) mice deficient in microglia [25] and may indicate a shift in immune cell composition in the lesions.Given the modest effect of the loss of IFNγ on survival and overall disease progression, we did not further explore this possibility in this study but cannot rule out that IFNγ loss has a small impact on CNS lesion content or size.

Anti-IFNγ therapies have been tested in multiple inflammatory
diseases with varying levels of success.The anti-IFNγ antibody emapalumab is used to treat patients with hemophagocytic lymphohistiocytosis (HLH) refractory to conventional therapy [26].HLH is characterised by uncontrolled proliferation of lymphocytes and macrophages that secrete high levels of inflammatory cytokines, including IFNγ.Anti-IFNγ therapy also has modest benefits in patients with Crohn's disease [27,28].Our findings support the notion that targeting IFNγ may provide some therapeutic benefits to patients with LS or other mitochondrial diseases, although it is clear that in the mouse model, targeting IFNγ alone is insufficient to reproduce the robust benefits of CSF1R inhibition.
The extension of survival and delay in disease progression provided by IFNγ loss provide additional data demonstrating that immune-mediated processes drive disease in the Ndufs4(À/À) model.However, the upstream signal initiating inflammation and production of IFNγ in LS remains unidentified, and the processes involved in macrophage activation and recruitment require further study.
Mice were weaned at P20-22 days of age.Ndufs4(À/À) animals were housed with control littermates for warmth as Ndufs4(À/À) mice have low body temperature [10].Mice were weighed and health assessed a minimum of three times a week.Following the onset of Ndufs4(À/À) symptoms, wet food was provided at the bottom of the cage.Animals were euthanized if they lost 20% of maximum body weight for two consecutive days, were immobile or were found moribund [12,13].Mice heterozygous for Ndufs4 have no reported phenotype, so controls consisted of both heterozygous and wild-type Ndufs4 animals.We refer to Ndfus4(Ctrl) mice here for clarity.The Ndufs4(Ctrl) and Ndufs4(À/À) mice wild type for Ifng or IP10 used in this study came from crosses of Ndufs4(+/À)/Ifng(+/À) mice, Ndufs4 (+/À)/IP10(+/À) mice and our general Ndufs4 colony.Mice were fed PicoLab Diet 5058 and were on a 12-h light-dark cycle.All animal experiments followed Seattle Children's Research Institute (SCRI) guidelines and were approved by the SCRI IACUC.
Clasping and ataxia were assessed by visual scoring and analysed as previously described [12].During disease progression, Ndufs4(À/À) animals can display intermittent/transient improvement of symptoms, so here we report whether the animal ever displayed the symptoms for two or more consecutive days.The onset of weight loss is indicated by the age of maximum body weight.
A Med Associates ENV-571M single-lane rotarod was used for the rotarod performance test.A mouse was placed on the rod already rotating at 6 rpm, and latency to fall was timed for a maximum of 600 s while rotation speed remained constant.For each mouse, three trials were performed with a minimum of 10 min between each trial.
The best of three trials was reported.

F I G U R E 3
Loss of Ifng delays the onset of weight loss and forelimb clasping in Ndufs4(À/À) mice.(A) The expression of IFNγ in stimulated and unstimulated splenocytes from Ndufs4(Ctrl) Ifng(+/+), Ifng(+/À) and Ifng(À/À) mice as determined by flow cytometry.Numbers in red indicate the mean fluorescence intensity (MFI) of cells in the upper right (IFNγ/CD8 double positive) quadrant and numbers in grey indicate the percent positive cells.(B) Mean fluorescence intensity of IFNγ staining in stimulated (+) and unstimulated (À) splenocytes of indicated genotype.