Society: AGA

Background
Aconitate decarboxylase 1 (ACOD1; also known as IRG-1) is the ubiquitous source of the metabolite itaconate, which dampens inflammasome activation by preventing HIF1α production of IL-1β and prevents TNF production through NRF2 activation. We reasoned that expression of ACOD1 is an inhibitory pathway within monocytes to curtail their inflammatory function, which could be manipulated in IBD therapy.

Methods
We performed single-cell RNA sequencing of monocytes from colonoscopic biopsies of treatment naïve IBD patients, as well as complementary analysis of DSS treated C57BL/6J (WT) mice, using single-cell protein-mRNA (Primeflow) technologies for validation. To determine the role of ACOD1 in colitis, Acod1^-/- mice were administered 2% DSS in drinking water for 6 days (acute colitis) or 4 days followed by 14 day recovery period (inflammation resolution) and assessed the colon myeloid compartment by multi-parameter flow cytometry. This was complemented with competitive bone marrow chimeric mice to assess the intrinsic role of ACOD1 controlling monocyte survival and proinflammatory status. Transcriptional profiling (Nanostring) was used to determine the molecular pathways influenced by ACOD1.

Results
We found that ACOD1/Acod1 represented an evolutionarily conserved of feature of inflammation-associated monocytes, with expression present in mucosal but not blood monocytes. Acod1^-/- mice demonstrated increased susceptibility to acute intestinal injury with DSS compared to co-housed WT controls, characterised by increased weight loss and colon shortening with significant accumulation of colonic neutrophils and CD64⁺Ly6C⁺ MHC-II^+/–monocytes. Consistent with Acod1 limiting inflammasome activation, Acod1^-/- monocytes demonstrated significantly greater IL-1b producing capabilities than WT controls.

To assess Acod1 deficient and sufficient haemopoietic cells in in the same environment, we generated competitive bone marrow chimeric mice. Acod1 deficiency conferred a significant advantage to monocyte recruitment to the colon, characterised by a 10fold increase in Ly6C⁺ MHC-II^–colon monocytes in colitis.

Transcriptional profiling of WT and Acod1^-/- colonic monocytes revealed significantly greater expression of molecules required for monocyte extravasation, including Sell (CD62L), Icam1, Ccr2 and Itgal (CD11a). In addition, expression of genes indicative of macrophage maturation such as Itgax (CD11c), Cd163 and Cx3cr1 were lower in the context of Acod1 deficiency, suggesting that normal maturation of these cells may be perturbed by loss of Acod1.

Conclusion
ACOD1/Acod1 represents an evolutionarily conserved, monocyte specific immuno-metabolic pathway that acts to limit monocyte recruitment, fate and pro-inflammatory function within the inflamed intestine.

% change in starting weight of Acod1-/- or C57BL6/JCrl (WT) mice after 6 days of 2% DSS (A) or 4 days plus 14 days normal drinking water (B). (<b>C</b>) Flow cytometry plots of colon lamina propria CD64<sup>+</sup> cells isolated from (<b>B</b>) with relative proportions of Ly6C<sup>+</sup> MHC-II<sup>-</sup> monocytes (P1) Ly6C<sup>+</sup> MHC-II<sup>+</sup> monocytes (P2) and Ly6C<sup>-</sup> MHC-II<sup>+</sup> macrophages (P3/4) and the proportion of IL-1β expressing P1 cells observed after 2hours incubation with 1ug/ml Momensin (<b>D</b>).Congenic CD45.1/CD45.2<sup>+</sup> mice were lethally irradiated and reconstitiuted with WT CD45.1 or <i>Acod1</i><sup>-/-</sup> CD45.2 BM, before treatment with 6 days 2% of DSS or normal drinking water. (<b>E</b>) Chimerism of colon CD64<sup>+</sup> cells relative to blood monocytes from gating strategy in (<b>C</b>). (<b>F</b>) CD45.1 and CD45.2 P1 and P2 moncytes from DSS treated mice were purified by flow cytometry, RNA extracted and profiled by Nanostring.

% change in starting weight of Acod1-/- or C57BL6/JCrl (WT) mice after 6 days of 2% DSS (A) or 4 days plus 14 days normal drinking water (B). (C) Flow cytometry plots of colon lamina propria CD64⁺ cells isolated from (B) with relative proportions of Ly6C⁺ MHC-II^- monocytes (P1) Ly6C⁺ MHC-II⁺ monocytes (P2) and Ly6C^- MHC-II⁺ macrophages (P3/4) and the proportion of IL-1β expressing P1 cells observed after 2hours incubation with 1ug/ml Momensin (D).Congenic CD45.1/CD45.2⁺ mice were lethally irradiated and reconstitiuted with WT CD45.1 or Acod1^-/- CD45.2 BM, before treatment with 6 days 2% of DSS or normal drinking water. (E) Chimerism of colon CD64⁺ cells relative to blood monocytes from gating strategy in (C). (F) CD45.1 and CD45.2 P1 and P2 moncytes from DSS treated mice were purified by flow cytometry, RNA extracted and profiled by Nanostring.

Adam Byrne

Appearances