A Truncated Form of HpARI Stabilizes IL-33, Amplifying Responses to the Cytokine

Abstract

The murine intestinal nematode Heligmosomoides polygyrus releases the H. polygyrus Alarmin Release Inhibitor (HpARI)-a protein which binds to IL-33 and to DNA, effectively tethering the cytokine in the nucleus of necrotic cells. Previous work showed that a non-natural truncation consisting of the first 2 domains of HpARI (HpARI_CCP1/2) retains binding to both DNA and IL-33, and inhibited IL-33 release in vivo. Here, we show that the affinity of HpARI_CCP1/2 for IL-33 is significantly lower than that of the full-length protein, and that HpARI_CCP1/2 lacks the ability to prevent interaction of IL-33 with its receptor. When HpARI_CCP1/2 was applied in vivo it potently amplified IL-33-dependent immune responses to Alternaria alternata allergen, Nippostrongylus brasiliensis infection and recombinant IL-33 injection, in direct contrast to the IL-33-suppressive effects of full-length HpARI. Mechanistically, we found that HpARI_CCP1/2 is able to bind to and stabilize IL-33, preventing its degradation and maintaining the cytokine in its active form. This study highlights the importance of IL-33 inactivation, the potential for IL-33 stabilization in vivo, and describes a new tool for IL-33 research.

IL-33 facilitates rapid expulsion of the parasitic nematode Strongyloides ratti from the intestine via ILC2- and IL-9-driven mast cell activation

“…Furthermore, HpARI binds to genomic DNA: this dual DNA/IL-33 binding allows HpARI to tether IL-33 within necrotic cells, preventing release of the cytokine [ 30 ]. The CCP1/2 truncation mutant of HpARI was shown to amplify rather than suppress IL-33 responses in vitro and in vivo , in a range of cytokine-, allergen-, and infection-driven systems [ 31 ]. CCP1/2 retains the ability to bind to DNA and IL-33, however cannot block the interaction of the cytokine with its receptor.…”

“…The HpARI and CCP1/2 proteins were produced in Expi293 cells and purified as previously described [ 30 , 31 ]. Briefly, Expi293 cells (ThermoFisher) were transfected with pSecTAG2A plasmids (Thermofisher Scientific) containing inserts encoding HpARI or CCP1/2 (with C-terminal Myc and 6-His tags), using Expifectamine transfection and enhancer reagents (Thermofisher Scientific) and following manufacturer´s instructions.…”

“…Here we employ the H. polygyrus-derived IL-33-suppressive alarmin release inhibitor (HpARI) [30] and an IL-33-stabilizing truncation variant of HpARI, CCP1/2 [31], to analyse the role of IL-33 during the early immune response to S. ratti. Neutralization of endogenous IL-33 increases intestinal parasite burdens while stabilization of endogenous IL-33 or application of recombinant (rec.)…”

“…Here we employ the H . polygyrus -derived IL-33-suppressive alarmin release inhibitor (HpARI) [ 30 ] and an IL-33-stabilizing truncation variant of HpARI, CCP1/2 [ 31 ], to analyse the role of IL-33 during the early immune response to S . ratti .…”

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“…The HpARI and CCP1/2 proteins were produced in Expi293 cells and purified as previously described [30,31]. Briefly, Expi293 cells (ThermoFisher) were transfected with pSecTAG2A plasmids (Thermofisher Scientific) containing inserts encoding HpARI or CCP1/2 (with C-…”