The Helicobacter cinaedi antigen CAIP participates in atherosclerotic inflammation by promoting the differentiation of macrophages in foam cells

Recent studies have shown that certain specific microbial infections participate in atherosclerosis by inducing inflammation and immune reactions, but how the pathogens implicated in this pathology trigger the host responses remains unknown. In this study we show that Helicobacter cinaedi (Hc) is a human pathogen linked to atherosclerosis development since at least 27% of sera from atherosclerotic patients specifically recognize a protein of the Hc proteome, that we named Cinaedi Atherosclerosis Inflammatory Protein (CAIP) (n = 71). CAIP appears to be implicated in this pathology because atheromatous plaques isolated from atherosclerotic patients are enriched in CAIP-specific T cells (10%) which, in turn, we show to drive a Th1 inflammation, an immunopathological response typically associated to atherosclerosis. Recombinant CAIP promotes the differentiation and maintenance of the pro-inflammatory profile of human macrophages and triggers the formation of foam cells, which are a hallmark of atherosclerosis. This study identifies CAIP as a relevant factor in atherosclerosis inflammation linked to Hc infection and suggests that preventing and eradicating Hc infection could reduce the incidence of atherosclerosis.

independent experiments repeated twice. Significance was determined by Student's t-test versus saline-exposed cells. ***p < 0.001. were challenged with 20 µg/ml CAIP or with 100 ng/ml E. coli LPS, as positive control. After 4 h, NF-κB activation (left panel) was measured using the luciferase reporter assay system, and normalized to that of saline-exposed cells, whose values were set at 1 A.U. IL-8 production (right panel) was quantified after 18 h stimulation with the specified agonists. Data are the mean values ± S.D. obtained from three independent experiments repeated twice. Significance was determined by Student's t-test versus saline-exposed cells. **p < 0.01; ***p < 0.001. (B) Monocytes were exposed to 20 µg/ml CAIP (boiled or not), to 100 ng/ml LPS (boiled or not) or saline (negative control) for 2 h and the expression of IL-12p40, IL-23p19, IL-1β, IL-6 and TNF-a was evaluated by RT-PCR. Data were normalized to an endogenous reference gene (GAPDH). Saline-treated cells were taken as reference and set as 1 A.U. (indicated by the dotted line) and the expression levels for treated cells were relative to the expression of negative control cells.

Supplementary Figure S4. CAIP and HP-NAP have a different impact on macrophages. (A)
Expression of CD86, CD163 and CD206 on macrophages exposed to CAIP or HP-NAP for 24 h.

Evaluation of the impact of LPS contamination on the cytokine expression induced in monocytes by CAIP
2×10 6 monocytes were exposed to 20 µg/ml CAIP (boiled or not), to 100 ng/ml LPS (boiled or not) or saline (negative control) for 2 h. Total RNA was extracted from 2×10 6  All datasets were indexed and integrated with software XDS 3 and merged and scaled with Scala 4 .

Characterization of the metal binding site of CAIP
CAIP dodecamer possesses four three-fold axes, each of them passing through the shell in two different 3-folds environments arranged as pores. One of the two three-fold type pores corresponds to the iron entry channel postulated for the other members of the family. The strongly hydrophilic and negatively charged character of this pore is fully conserved with respect to HP-NAP. On the contrary, the other pore type is smaller and less negatively charged.
A Fe ion was fitted in the site occupied by the iron in the other members of the family, for a total of 12 iron sites. These cations represent the ferroxidase centres, where Fe 2+ from the environment is internalized and oxidized, before being stored in the inner cavity. The ion present in CAIP displays a distorted tetrahedral coordination, where three corners are occupied by protein atoms, two oxygens and one nitrogen. They derive from the side chains of Asp51, Glu55 from a subunit and His24 from a nearby one. The fourth coordination position is occupied by a single atom that we interpreted as a solvent molecule. Nevertheless, its mean distance to the Fe ion (around 2.6 Å-2.8 Å) and the fact that some residual density is present in the Fourier-difference map suggest that a heavier atom is present in this position. Other Dps-like proteins contains a di-iron site, but in this case this putative second cation does not present any coordination, being only linked to the Fe ion.