Additional file 1 of Interleukin-6-elicited chronic neuroinflammation may decrease survival but is not sufficient to drive disease progression in a mouse model of Leigh syndrome

Additional file 1: Supplementary Figure 1. Microgliosis and astrogliosis at the late stage divided by sex. Related with Figures 4 and 5. (a) Quantification of IBA-1 fluorescence intensity in the VN, cerebellum, OB, cortex, and CA1, as well as the number of IBA-1+ cells in the cortex, and CA1 of the different genotypes. (b)GFAP mean fluorescence intensity quantification in the VN, cerebellum, OB, cortex, and CA1. ● Ndufs4 effect p≤ 0.05,  ★ GF-IL6 effect p≤ 0.05, ♦ p≤ 0.05 interaction between both factors. Supplementary Figure 2. Microgliosis and astrogliosis at the mid stage divided by sex. Related with Figure 6. (a) Quantification of IBA-1 fluorescence intensity in the VN, cerebellum, OB, cortex, and CA1, as well as the number of IBA-1+ cells in the cortex, and CA1 of the different genotypes. (b) GFAP mean fluorescence intensity quantification in the VN, cerebellum, OB, cortex, and CA1. ● Ndufs4 effect p≤ 0.05,  ★ GF-IL6 effect p≤ 0.05, ♦ p≤ 0.05 interaction between both factors

Primer sequences used.

<p>Primer sequences used.</p

Indirect calorimetric parameters during acute exercise.

<p>The treadmill was initially set to a constant speed (0.15 m/s) for 2 min, increased at 0.2 m/s for 2 min, and then acceleration was increased by 0.05 m/s every two minutes. Although treadmill speed significantly affects VO₂, EE and RER in both genders (<b>A-C</b>), only in males a significant effect of muscle IL-6 deficiency in RER (<b>C</b>) was noted. ★<i>p</i> < 0.05 <i>versus</i> floxed mice. ◆ <i>p</i> at least < 0.05 <i>versus</i> male mice.</p

Body weight changes following fasting and refeeding.

<p>Adaptation to isolation measured by body weight gain in floxed and <i>muscle IL-6 KO</i> male (n = 13/12) and female (n = 14/14) mice (<b>A</b>). Female mice recovered somewhat faster but no effects of genotype were obvious. Following this period of acclimatization, the mice were subjected to fasting overnight and then food was allowed (refeeding) (<b>B, C</b>). Body weight changes following fasting and at 4, 24 and 48h after refeeding (<b>B</b>) and food intake at 1, 2, 3, 4, 24, and 48h after refeeding (<b>C</b>) are shown. The effect of muscle IL-6 deficiency was significant only for food intake and in some of the refeeding periods in female mice. ★ <i>p</i> at least < 0.05 <i>versus</i> floxed mice.</p

Effect of leptin treatment in WAT, BAT and liver weight.

<p>Gonadal, subcutaneous and brown fat depots and liver were weighed after leptin / vehicle treatment in males and females, and are expressed as the percentage of body weight. Female mice showed decreased depots of gonadal and brown fat <i>per se</i>. Additionally, subcutaneous fat depots were affected by leptin treatment in a gender specific manner, since in males, muscle IL-6 absence caused a higher loss of subcutaneous fat depots in response to leptin (34%). ▲and ★ <i>p</i> at least < 0.05 <i>versus</i> vehicle and floxed mice, respectively. ◆ <i>p</i> at least < 0.05 <i>versus</i> male mice.</p

Analysis of plasma triglycerides, cholesterol and glucose levels after leptin treatment.

<p>Female mice showed decreased triglycerides and cholesterol levels <i>per se</i>. Leptin treatment significantly decreased triglycerides and glucose plasma levels in both genders, with no effect of muscle IL-6 deficiency. In contrast, leptin decreased cholesterol levels only in female mice, and muscle IL-6 deficiency increased serum cholesterol regardless of the treatment (vehicle or leptin) in male mice. ▲and ★ <i>p</i> at least < 0.05 <i>versus</i> vehicle and floxed mice, respectively. ◆ <i>p</i> at least < 0.05 <i>versus</i> male mice.</p

Deficiency of muscle IL-6 is not associated with changes in the muscle or liver insulin-signalling pathway.

<p><b>A</b>: Representative western blot images of P-Akt/Akt in tibialis, soleus and liver from males and females, floxed and <i>muscle IL-6 KO</i> after 30 min of insulin administration (i.p. 1 UI/animal) or vehicle in 4 hours-starved mice. <b>B</b>: Quantification of the northern blots. ▲ <i>p</i> < 0.001 <i>versus</i> vehicle.</p

Core temperature in the light phase.

<p>Core temperature in floxed and <i>muscle-IL6 KO</i> male (n = 11/10), and female (n = 9/10) mice. Absence of muscle IL-6 determined a lower core temperature in male but not female mice in the light phase. ★ <i>p</i> at least < 0.05 <i>versus</i> floxed mice.</p

Gene expression in tibialis muscle, white adipose tissue and brown adipose tissue after leptin treatment.

<p>As expected, <i>muscle IL-6 KO</i> mice showed decreased IL-6 mRNA levels in muscle but not in other tissues. This muscle IL-6 deficiency did not affect the expression of the other genes in muscle; in contrast, it did affect that of Fabp3 in WAT. Sex was a clear factor influencing the expression of some of these genes, as was leptin injection. Interestingly, sex also modified the effect of leptin in WAT and BAT in some cases, inverting the effect of the hormone. ▲and ★ <i>p</i> at least < 0.05 <i>versus</i> vehicle and floxed mice, respectively. Δ denotes a significant interaction between sex and leptin <i>p</i> at least < 0.05. ◆ <i>p</i> at least < 0.05 <i>versus</i> male mice. ☆ denotes a significant interaction between sex and genotype <i>p</i> at least < 0.05.</p

Deficiency of muscle IL-6 influences the response to restraint stress.

<p><b>A</b>: Sixty min of restraint stress prominently increased serum corticosterone levels, which returned toward normal after 120 min of stress recovery. A sex-dependent effect of muscle IL-6-deficiency was present, since the stress response was lower in female but not male <i>muscle IL-6 KO</i> mice. <b>B</b>: Adrenal StAR levels in the adrenal glands were also evaluated by western blot. At the top the blot obtained for female mice is shown. At the bottom, the quantification carried out for both genders revealed increased adrenal StAR in females of <i>muscle IL-6 KO</i> after 120 min of stress recovery. ★ <i>p</i> at least < 0.05 <i>versus</i> floxed mice.</p