Characterisation of the secreted apyrase family of Heligmosomoides polygyrus

Apyrases are a recurrent feature of secretomes from numerous species of parasitic nematodes. Here we characterise the five apyrases secreted by Heligmosomoides polygyrus, a natural parasite of mice and a widely used laboratory model for intestinal nematode infection. All five enzymes are closely related to soluble calcium-activated nucleotidases described in a variety of organisms, and distinct from the CD39 family of ecto-nucleotidases. Expression is maximal in adult worms and restricted to adults and L4s. Recombinant apyrases were produced and purified from Pichia pastoris. The five enzymes showed very similar biochemical properties, with strict calcium dependence and a broad substrate specificity, catalysing the hydrolysis of all nucleoside tri- and diphosphates, with no activity against nucleoside monophosphates. Natural infection of mice provoked very low antibodies to any enzyme, but immunisation with an apyrase cocktail showed partial protection against reinfection, with reduced egg output and parasite recovery. The most likely role for nematode secreted apyrases is hydrolysis of extracellular ATP, which acts as an alarmin for cellular release of IL-33 and initiation of type 2 immunity

Standing economy: does the heterogeneity in the energy cost of posture maintenance reside in differential patterns of spontaneous weight-shifting?

Due to sedentarity-associated disease risks, there is much interest in methods to increase low-intensity physical activity. In this context, it is widely assumed that altering posture allocation can modify energy expenditure (EE) to impact body-weight regulation and health. However, we have recently shown the existence of two distinct phenotypes pertaining to the energy cost of standing-with most individuals having no sustained increase in EE during steady-state standing relative to sitting comfortably. Here, we investigated whether these distinct phenotypes are related to the presence/absence of spontaneous "weight-shifting", i.e. the redistribution of body-weight from one foot to the other. Using indirect calorimetry to measure EE in young adults during sitting and 10 min of steady-state standing, we examined: (i) heterogeneity in EE during standing (n = 36); (ii) EE and spontaneous weight-shifting patterns (n = 18); (iii) EE during spontaneous weight-shifting versus experimentally induced weight-shifting (n = 7), and; (iv) EE during spontaneous weight-shifting versus intermittent leg/body displacement (n = 6). Despite heterogeneity in EE response to steady-state standing, no differences were found in the amount or pattern of spontaneous weight-shifting between the two phenotypes. Whilst experimentally induced weight-shifting resulted in a mean EE increase of only 11% (range: 0-25%), intermittent leg/body displacement increased EE to >1.5 METs in all participants. Although the variability in spontaneous weight-shifting signatures between individuals does not appear to underlie heterogeneity in the energy cost of standing posture maintenance, these studies underscore the fact that leg/body displacement, rather than standing posture alone, is needed to increase EE above the currently defined sedentary threshold

Differential regulation of allergic airway inflammation by acetylcholine

Acetylcholine (ACh) from neuronal and non-neuronal sources plays important roles in the regulation of immune responses and is associated with the development of several disease pathologies. We have previously demonstrated that group 2 innate lymphoid cell (ILC2)-derived ACh is required for optimal type 2 responses to parasitic infection, and therefore sought to determine whether this also plays a role in allergic inflammation. RoraCre+ChatLoxP mice (in which ILC2s cannot synthesize ACh) were exposed to an allergenic extract of the fungus Alternaria alternata, and immune responses in the airways and lung tissues analysed. Airway neutrophilia and expression of the neutrophil chemoattractants CXCL1 and CXCL2 were enhanced 24 hours after exposure, suggesting that ILC2-derived ACh plays a role in limiting excessive pulmonary neutrophilic inflammation. The effect of non-selective depletion of ACh was examined by intranasal administration of a stable parasite-secreted acetylcholinesterase. Depletion of airway ACh in this manner resulted in more profound enhancement of neutrophilia and chemokine expression, suggesting multiple cellular sources for release of ACh. In contrast, depletion of ACh inhibited Alternaria-induced activation of ILC2s, suppressing expression of IL-5, IL-13 and subsequent eosinophilia. Depletion of ACh resulted in a reduction of macrophages with an alternatively activated M2 phenotype, and an increase in M1 macrophage marker expression. These data suggest that ACh regulates allergic airway inflammation in several ways, enhancing ILC2-driven eosinophilia, but suppressing neutrophilia through reduced chemokine expression

Acetylcholine production by group 2 innate lymphoid cells promotes mucosal immunity to helminths

Innate lymphoid cells (ILCs) are critical mediators of immunological and physiological responses at mucosal barrier sites. Whereas neurotransmitters can stimulate ILCs, the synthesis of small-molecule neurotransmitters by these cells has only recently been appreciated. Group 2 ILCs (ILC2s) are shown here to synthesize and release acetylcholine (ACh) during parasitic nematode infection. The cholinergic phenotype of pulmonary ILC2s was associated with their activation state, could be induced by in vivo exposure to extracts of Alternaria alternata or the alarmin cytokines interleukin-33 (IL-33) and IL-25, and was augmented by IL-2 in vitro. Genetic disruption of ACh synthesis by murine ILC2s resulted in increased parasite burdens, lower numbers of ILC2s, and reduced lung and gut barrier responses to Nippostrongylus brasiliensis infection. These data demonstrate a functional role for ILC2-derived ACh in the expansion of ILC2s for maximal induction of type 2 immunity