Malnutrition promotes prostaglandin over leukotriene production and dysregulates eicosanoid-cytokine crosstalk in activated resident macrophages

Abstract

We previously described a murine model of malnutrition that mimicked features of moderate human malnutrition, and led to increased dissemination of Leishmania donovani. In this study, we investigated the effect of malnutrition on macrophage production of cytokines, prostaglandins (PGs), and leukotrienes (LTs). Using either LPS or calcium ionophore A23187 as a stimulus, macrophages from the malnourished mice produced a 3-fold higher PG/LT ((PGE₂+6-keto-PGF_1α)/(LTB₄+cysteinyl leukotrienes)) ratio than macrophages from well-nourished mice. LPS-stimulated macrophages from the malnourished mice produced decreased levels of TNF-α, GM-CSF, and IL-10, but similar levels of IL-6 and NO compared to well-nourished mice. A complex crosstalk between the eicosanoids and cytokines in the LPS-stimulated macrophages from the malnourished mice was evident by the following: (1) high levels of PG secretion despite low levels of TNF-α; (2) supplemental IL-10 modulated the excessive PG production; (3) GM-CSF rectified the PG/LT ratio, but did not correct the abnormal cytokine profile; and (4) inhibitors of cyclooxygenase decreased the PG/LT ratio, but did not affect TNF-α. Thus, in this model of malnutrition, there is a relative increase in anti-inflammatory PGs compared to pro-inflammatory LTs, which may contribute to immunodeficiency.

Introduction

In its synergy with infection, malnutrition contributes to 56% of all childhood deaths worldwide [1]. Previously, we have described a murine model of multinutrient undernutrition that mimics human weanling malnutrition [2]. In this model, we have shown that malnutrition leads to increased early dissemination of the intracellular parasite Leishmania donovani [2] and causes dysregulation of the macrophage pro-inflammatory cytokine network and nuclear factor kappa-B activation [3].

In addition to cytokines, lipid mediators such as eicosanoids (20-carbon molecules generated from arachidonic acid (AA)), also play an important role in inflammation. Two classes of eicosanoids important in the early inflammatory response are prostaglandins (PGs, a family of unsaturated polyoxygenated fatty acids) and leukotrienes (LTs, oxygenated fatty acids containing a conjugated triene). The initial step in prostaglandin production is the conversion of AA into PGG₂ by cyclooxygenase. There are two distinct cyclooxygenase isoforms: COX-2 (induced by various inflammatory stimuli) and COX-1 (primarily constitutive; partially inducible). PGG₂ is peroxidized to PGH₂, which is then converted to various prostanoids, including PGE₂ and PGI₂ (prostacyclin), the two most abundant PGs in the murine system [4]. PGE₂ and PGI₂ share some biological activities because both increase intracellular levels of cyclic adenosine monophosphate (cAMP) [5], [6].

The first step in the biosynthesis of leukotrienes is the conversion of AA to LTA₄ by 5-lipoxygenase (5-LO). Leukotriene A₄ can be converted to LTB₄ or conjugated with glutathione to yield LTC₄. The latter is further converted to LTD₄ and LTE₄ by successive elimination of glutamyl and glycine residues. Leukotriene C₄, LTD₄, and LTE₄, are collectively called cysteinyl leukotrienes (cysLTs). The LTs decrease levels of cAMP and increase levels of cyclic guanosine monophosphate (cGMP) [7], [8]. In general, the LTs are pro-inflammatory [9], whereas the PGs are anti-inflammatory [10].

Previously, we have shown in a murine model that malnutrition results in increased PGE₂ release by cells from the local draining lymph node after cutaneous infection with L. donovani and that PGE₂ levels correlate with the degree of parasite dissemination [2]. PGE₂ has been implicated as an immunosuppressive mediator in other infections as well: cutaneous leishmaniasis [11], Mycobacterium avium infections [12], tuberculosis [13], leprosy [14], cysticercosis [15], influenza [16], and amebiasis [17]. Conversely, the LTs contribute to host defense in bacterial pneumonia [18], vesicular stomatitis virus encephalitis [19], histoplasmosis [20], schistosomiasis [21], Chagas’ disease [22], mycobacterial infections [23], cytomegalovirus infection [24], and toxoplasmosis [25].

Increased PGE₂ production has been observed in various models of malnutrition [26], [27], [28], [29]. Skerrett reported that alveolar macrophages from protein and calorie-deficient rats had increased PGE₂ and decreased LTB₄ release after Listeria infection [28]. Lower levels of neutrophil LTC₄ release have been reported in the malnourished elderly patients [30].

Both PGs and LTs are derived from AA and thus there may be a partition of the common substrate between these two enzymatic pathways. PGs and LTs counter-regulate each other and these two classes of eicosanoids have many opposing physiologic and cellular effects [31]. The limited previous work on eicosanoid regulation during malnutrition has not adequately addressed the relationship between these two classes of eicosanoids and the inter-relationship of these lipid mediators with the cytokine network. We hypothesized that malnutrition would produce an increase in immunosuppressive PGs relative to the pro-inflammatory LTs. To test this hypothesis, we studied the effect of multinutrient malnutrition on eicosanoid release from stimulated murine resident macrophages using two prototypical stimuli, lipopolysaccharide (LPS) and a calcium ionophore. In various macrophage systems, eicosanoid production is regulated by the IL-10, TNF-α, and GM-CSF [32], [33], [34], and therefore we investigated the relationship of eicosanoid levels to these cytokines in this model. Also, we investigated the hypothesis that inhibition of cyclooxygenase would decrease the abnormal level of PGs relative to the LTs.