Alpha-melanocyte-stimulating hormone contributes to an anti-inflammatory response to lipopolysaccharide

Abstract

Objective: During infection, metabolism and immunity react dynamically to promote survival through mechanisms that remain unclear. Pro-opiomelanocortin (POMC) cleavage products are produced and released in the brain and in the pituitary gland. One POMC cleavage product, alpha-melanocyte-stimulating hormone (α-MSH), is known to regulate food intake and energy expenditure and has anti-inflammatory effects. However, it is not known whether α-MSH is required to regulate physiological anti-inflammatory responses. We recently developed a novel mouse model with a targeted mutation in Pomc (Pomc^tm1/tm1 mice) to block production of all α-MSH forms which are required to regulate metabolism. To test whether endogenous α-MSH is required to regulate immune responses, we compared acute bacterial lipopolysaccharide (LPS)-induced inflammation between Pomc^tm1/tm1 and wild-type Pomc^wt/wt mice.

Methods: We challenged 10- to 14-week-old male Pomc^tm1/tm1 and Pomc^wt/wt mice with single i.p. injections of either saline or low-dose LPS (100 μg/kg) and monitored immune and metabolic responses. We used telemetry to measure core body temperature (T_b), ELISA to measure circulating cytokines, corticosterone and α-MSH, and metabolic chambers to measure body weight, food intake, activity, and respiration. We also developed a mass spectrometry method to measure three forms of α-MSH produced in the mouse hypothalamus and pituitary gland.

Results: LPS induced an exaggerated immune response in Pomc^tm1/tm1 compared to Pomc^wt/wt mice. Both groups of mice were hypoactive and hypothermic following LPS administration, but Pomc^tm1/tm1 mice were significantly more hypothermic compared to control mice injected with LPS. Pomc^tm1/tm1 mice also had reduced oxygen consumption and impaired metabolic responses to LPS compared to controls. Pomc^tm1/tm1 mice had increased levels of key proinflammatory cytokines at 2 h and 4 h post LPS injection compared to Pomc^wt/wt mice. Lastly, Pomc^wt/wt mice injected with LPS compared to saline had increased total α-MSH in circulation 2 h post injection.

Conclusions: Our data indicate endogenous α-MSH contributes to the inflammatory immune responses triggered by low-dose LPS administration and suggest that targeting the melanocortin system could be a potential therapeutic for the treatment of sepsis or inflammatory disease.

Keywords: LPS; Mouse model; POMC; Thermogenesis; α-MSH.

Figure 1

LPS reduced activity and core body temperature in Pomc^wt/wtand Pomc^tm1/tm1mice.Pomc^wt/wt and Pomc^tm1/tm1 mice were injected (i.p.) with saline on Day 1 (A) and 100 μg/kg LPS on Day 2 (B) 1 h before lights off, and relative activity and core body temperature was recorded by telemeter every 10 min. Activity data is binned in 1 h blocks. (C) Summary figure of all 10 min recordings for Days 1 and 2 superimposed. Core body temperature was also recorded after i.p. injections of saline on Day 1 (D) and 100 μg/kg LPS on Day 2 (E). Summary data are shown for Days 1 and 2 superimposed (F). The data are expressed as the mean ± SEM. ∗p < 0.05 compared to littermate controls.

Figure 2

Pomc^tm1/tm1mice had reduced oxygen consumption and RER after LPS injection. (A–D)Pomc^wt/wt and Pomc^tm1/tm1 mice were injected (i.p.) with saline 1 h before lights off on Day 1. VO₂ was measured and RER calculated for 3 days. (E–H)Pomc^wt/wt and Pomc^tm1/tm1 mice were injected i.p. with 100 μg/kg LPS. Gray boxes indicate dark cycle, and bar graphs show average values per mouse for the entire dark period after saline or LPS injection (dotted line region on the line graphs). The data are expressed as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01 compared to littermate controls.

Figure 3

Peripheral α-MSH increased in Pomc^wt/wtmice and Pomc^tm1/tm1mice had normal corticosteroid production but increased leptin after LPS injection.Pomc^wt/wt and Pomc^tm1/tm1 mice were injected with 100 μg/kg LPS and plasma was collected 2 h later. (A) Total α-MSH (all 3 forms combined) was measured by ELISA in Pomc^wt/wt mice. (B) Corticosterone and (C) leptin were measured in Pomc^wt/wt and Pomc^tm1/tm1 mice by ELISA. The data are expressed as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01 compared to littermate controls.

Figure 4

Pomc^tm1/tm1mice had increased cytokine levels compared to Pomc^wt/wtmice 2 h and 4 h after LPS injection. Plasma from Pomc^wt/wt and Pomc^tm1/tm1 mice were collected 2 h (A) and 4 h (B) after 100 μg/kg i.p. LPS injection and cytokine levels were quantified using a cytokine multiplex assay. The data are expressed as the mean ± SEM. ∗p < 0.05 and ∗∗p < 0.01 compared to littermate controls.