Fasting-induced hypoleptinemia expands functional regulatory T cells in systemic lupus erythematosus

Abstract

Fasting is beneficial in the prevention and amelioration of the clinical manifestations of autoimmune diseases including systemic lupus erythematosus (SLE). The mechanisms responsible for these effects are not well understood. During fasting, there is a dramatic reduction of the levels of circulating leptin, an adipokine with proinflammatory effects. Leptin also inhibits CD4⁺CD25⁺Foxp3⁺ regulatory T cells (T_Reg), which are known to contribute significantly to the mechanisms of peripheral immune tolerance. Here we show that fasting-induced hypoleptinemia in (NZB×NZW)F₁ lupus-prone mice induced an expansion of functional T_Reg that was reversed by leptin replacement. The specificity of the findings was indicated by the lack of these effects in leptin-deficient ob/ob mice and in leptin receptor-deficient db/db mice. These observations help to explain the beneficial effects of fasting in autoimmunity and could be exploited for leptin-based immune intervention in SLE.

Introduction

Fasting and/or caloric restriction are known to alleviate the manifestations of autoimmune diseases through mechanisms that remain mostly elusive (1). For example, caloric restriction prolongs life in (NZB×NZF)F₁ (NZB/W) mice (2) that spontaneously develop systemic lupus erythematosus (SLE), an autoimmune disease characterized by the breakdown of tolerance to multiple nuclear antigens and an uncontrolled activation of self-reactive lymphocytes that cause inflammation and tissue damage. Although it has been shown that in NZB/W mice the reduction of caloric intake associates with a decrease in lymphoproliferation, antibody production and secretion of pro-inflammatory mediators (3), it remains unclear what drives the observed beneficial clinical, immunological and biochemical effects (4). During fasting, the levels of the circulating adipokine leptin are dramatically reduced (5). Leptin has all the characteristics of a pro-inflammatory cytokine (6) and links nutritional status with neuroendocrine functions and immune responses. Leptin promotes T helper (Th)1 cell differentiation and the development of autoimmune responses in several animal disease models (7), and it can act as a negative factor for the expansion of regulatory CD4⁺ T cells (T_Reg) - a subset of T cells with an important role in the maintenance of peripheral immune tolerance to self antigens (8).

In an attempt to connect these observations, we investigated the effects of fasting in NZB/W mice and found that the reduction in circulating levels of leptin caused by starvation directly promoted the expansion of functional T_Reg. These findings can help to explain some of the beneficial effects of fasting in SLE.

Materials and Methods

Mice

C57BL6/J (B6) wild-type (WT) mice, leptin-deficient B6^ob/ob (ob/ob), leptin receptor-deficient B6^db/db (db/db), and NZB/W mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained at the University of California Los Angeles (UCLA). Mice were treated in accordance with institutional guidelines under approved protocols. All experiments were conducted in age-matched female mice that were divided into three groups. One control group had ad libitum access to food and received intraperitoneal injections of 0.2 mL PBS at 9 AM and 6 PM daily for two days. The other two groups of mice were deprived of food for 48 hours and received intraperitoneal injections of either 0.2 mL PBS or recombinant leptin (R&D Systems, Minneapolis, MN) dissolved in PBS at a dose of 1 µg/g of body weight twice daily (also at 9 AM and 6 PM). All mice had continuous access to water.

Cell isolation and staining

After blood drawing, erythrocytes were removed using red cell lysing buffer (Sigma-Aldrich, St. Louis, MO) and PBMC were used for flow cytometry. For splenocytes, single-cell suspensions were prepared by passing cells through a cell strainer before red cell lysis and resuspension in HL-1 medium (Bio Whittaker,Walkersville, MD). CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were isolated via magnetic bead separation with Miltenyi Biotec kits using an AutoMACS System (Miltenyi Biotec, Auburn, CA) and were found >95% pure by flow cytometry analysis.

Flow cytometry

Phenotypic analyses were performed with combinations of fluorochrome-conjugated Ab using standard techniques. After Fc blocking, fluorochrome-conjugated anti-mouse Ab to CD4 and CD25 (eBioscience, San Diego, CA) or isotype control Ab were used for staining prior to acquisition on a FACSCalibur™ flow cytometer (BD Biosciences, San Jose, CA) and subsequent analysis using FloJo software (Tree Star Inc., Ashland, OR). For intracellular staining, cells were first stained for the expression of cell surface markers and then fixed, permeabilized, and stained using the Foxp3 staining kit (eBioscience) according to the manufacturer’s instructions.

Suppression assays

After cell isolation, CD4⁺CD25⁻ T cells (T_Eff) were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) by incubation in 0.5 µM CFSE at 37°C for 10 min. Samples were then placed in HL-1/5% FCS and incubated in ice for 5 minutes, then washed 2 times. T_Reg: T_Eff (ratio 1:1 or 1:4) mixtures or control samples consisting of T_Eff alone were plated in 96-well round bottom plates (Corning Life Sciences, Lowell, MA). All wells contained an equal number of CD4⁺ T cells. Dynabeads mouse anti-CD3/CD28 beads were added to a final ratio of 0.5 bead per cell. Samples were incubated in a 37°C incubator for 72 hr. Proliferation of CFSE-labeled T_Eff was evaluated by flow cytometry and expressed as FlowJo software-calculated division index (the average number of cell divisions). Supernatants were collected for ELISA detection of IFNγ in the cocultures. Percent suppression was calculated using proliferation measurements and the following formula: % suppression = (1 − T_Reg: T_Eff/T_Eff alone) × 100.

ELISA

Concentration of leptin and IFNγ were determined by commercial ELISA kits (R&D Systems) according to the manufacturer’s instructions.

Statistical analyses

Analyses were performed using Prizm 4 software (GraphPad, San Diego, CA). Nonparametric testing among three groups was performed by Kruskal-Wallis ANOVA. Comparisons among three groups were performed by Dunn’s multiple comparison test. P values <0.05 were considered statistically significant.

Results and Discussion

The effects of fasting were compared among wild type (WT) mice, leptin-deficient ob/ob mice, leptin receptor-deficient db/db mice, and lupus prone NZB/W mice. Control animals were fed ad libitum or starved 48 hours (treated or not with leptin). As expected, starvation led to a reduction in body weight in all groups of mice, when compared to ad libitum-fed animals (Table I). Starvation also associated with a significant reduction in the number of splenocytes and T_Eff in starved WT, ob/ob and NZB/W mice (Supplemental Table I).

Table I.

Mice		ad libitum	fast + PBS	fast + leptin
WT	initial body weight (g)	20.59 ± 0.72	20.97 ± 0.79	20.59 ± 0.74
	body weight after 48 h (g)	20.77 ± 0.68	17.20 ± 0.70a	17.8 ± 0.78a
	plasma leptin (ng/ml)	7.22 ± 0.67	4.06 ± 0.64b	6.78 ± 0.55c
ob/ob	initial body weight (g)	58.36 ± 0.61	58.42 ± 1.39	58.66 ± 1.38
	body weight after 48 h (g)	58.46 ± 0.60	54.82 ± 1.47	54.68 ± 1.36
	plasma leptin (ng/ml)	0.19 ± 0.01	0.23 ± 0.01	6.35 ± 0.73d
db/db	initial body weight (g)	59.5 ± 0.29	57.1 ± 1.88	60.77 ± 1.36
	body weight after 48 h (g)	59.6 ± 0.25	52.9 ± 2.89	56.13 ± 1.72
	plasma leptin (ng/ml)	9.43 ± 1.13	8.01 ± 0.29	11.46 ± 0.65c
NZB/W	initial body weight (g)	23.82 ± 0.45	23.63 ± 0.35	22.92 ± 0.38
	body weight after 48 h (g)	23.87 ± 0.42	19.92 ± 0.58e	19.30 ± 0.43e
	plasma leptin (ng/ml)	9.79 ± 0.40	5.75 ± 0.48f	8.70 ± 0.58c

Note – Mice were fed ad libitum or fasted 48 hours. Values represent mean±SEM.

^aP<0.05 vs. initial body weight;

^bP<0.05 vs. ad libitum;

^cP<0.05 vs. fast + PBS;

^dP<0.01 vs. ad libitum and fast + PBS;

^eP<0.001 vs. initial body weight;

^fP<0.01 vs. ad libitum.

It is well established that fasting associates with the reduction of circulating leptin levels (5). Accordingly, WT and NZB/W mice starved for 48 hours had hypoleptinemia when compared to ad libitum-fed animals, whereas no changes in leptin levels were observed in starved ob/ob mice (which cannot produce functional leptin because of a mutation in the leptin gene [9]) and db/db mice (which carry a mutation in the leptin receptor [10]) (Table I). The administration of leptin to starved mice restored the circulating levels of this adipokine to normal concentrations in leptin-sufficient mice (Table I), and spleen cellularity returned to values similar to those seen in ad libitum-fed controls in starved mice that received leptin (Supplemental Table I).

Since we reported that leptin can constrain the expansion of T_Reg in vivo (8), we investigated whether the reduction in circulating levels of leptin induced by fasting could affect the T_Reg numbers. It was found that the frequency of T_Reg in starved WT and NZB/W lupus mice was significantly increased as compared to ad libitum–fed mice (Fig. 1). Of interest, leptin administration to ob/ob mice, which had no change of leptin levels after starvation (Table I), associated with a reduction in the number of T_Reg (Fig. 1c–d), suggesting that leptin per se has inhibitory effects on the expansion of T_Reg in vivo. In confirmation of these findings, db/db mice (which are hyperleptinemic but unresponsive to leptin) showed no change in T_Reg frequency after starvation, and leptin replacement did not change this finding (Fig. 1e–f). Moreover, T_Reg that had expanded after starvation had similar CD25 expression (which can be a marker of immune cell activation) in the different groups of animals (Supplemental Table I). Finally, the frequency of CD4⁺CD25⁻ cells (T_Eff) in PBMC was reduced by fasting and restored by leptin replacement in WT, ob/ob and NZB/W mice (Supplemental Table I).

Figure 1.

Fasting expands T_Reg in NZB/W lupus mice. CD4⁺CD25⁺Foxp3⁺ T cells from WT, ob/ob, db/db, and NZB/W mice fed ad libitum or starved 48 hours, without or with leptin treatment. Representative (a, c, e, g) and cumulative flow cytometry data (b, d, f, h) (n = 4–l7 per group) on gated CD4⁺ T cells from PBMC in one of three independent experiments. *P<0.05; **P<0.01.

Taken together, these results indicate that fasting associates with an increase in T_Reg frequency when the leptin pathway is intact (in WT and NZB/W mice), and the T_Reg frequency remains unaltered when fasting cannot alter leptinemia due to an impairment of the leptin/leptin receptor axis (such as in ob/ob and in db/db mice). These events depend on leptin because in WT and NZB/W mice the T_Reg frequency returned to numbers comparable to those found in ad libitum-fed mice following leptin replacement (Fig. 1).

To also address whether the observed changes in T_Reg frequency associated or not with a normal function of the suppressor cells, in vitro suppression assays were performed. The results showed that the changes in T_Reg frequency under the different conditions (ad libitum, fast and fast + leptin) did not associate with differences in the suppressive capacity of T_Reg on T_Eff (Fig. 2 and Supplemental Table II), indicating that T_Reg expanded by fasting were functionally comparable among groups.

Figure 2.

In vitro suppression of T_Eff proliferation by T_Reg (1:1 ratio) in ad libitum-fed vs. 48 hours-starved mice without or with leptin replacement. a, WT mice; c, ob/ob, e, db/db; g, NZB/W mice. The figure shows proliferation in flow cytometry of anti-CD3/CD28 Ab-stimulated, splenocyte-derived, CFSE-labeled T_Eff cultured alone (grey line) or with (black line) T_Reg.

Altogether, these results widen earlier observations that showed that caloric restriction ameliorated SLE manifestations (2, 11–12). The observation that functional T_Reg could expand during fasting in lupus mice is relevant to the disease pathogenesis because the frequency of T_Reg is lower in NZB/W mice as compared to WT mice (13–14), and this aspect has been linked to an inability of the dysfunctional immune system in SLE to control disease course and progression (15).

The link between nutritional status and immune regulation that has been suggested by recent reports showing that the mTOR pathway could influence the activity of T_Reg through leptin (16), identifies in the current study a new possibility to modulate T_Reg activity in SLE via leptin-based intervention (also considering that leptin is elevated in SLE patients [17]). In particular, the frequency of T_Reg could be tuned for therapeutic purposes in SLE not only through fasting but also, more specifically, using neutralizing antibodies to leptin, to promote the expansion in vivo of functional T_Reg in the disease.

Abbreviations

SLE

systemic lupus erythematous

T_Reg

regulatory CD4⁺ T cells

T_Eff

effector CD4⁺ T cells