Cody Diehl’s research while affiliated with Brigham Young University - Idaho and other places

In this Letter, affiliation number 1 was originally missing from the HTML; the affiliations were missing for author Ming-Yow Hung in the HTML; and the Fig. 4 legend erroneously referred to panels a-h, instead of a-g. These errors have been corrected online.

Oxidized phospholipids (OxPL) are ubiquitous, are formed in many inflammatory tissues, including atherosclerotic lesions, and frequently mediate proinflammatory changes 1 . Because OxPL are mostly the products of non-enzymatic lipid peroxidation, mechanisms to specifically neutralize them are unavailable and their roles in vivo are largely unknown. We previously cloned the IgM natural antibody E06, which binds to the phosphocholine headgroup of OxPL, and blocks the uptake of oxidized low-density lipoprotein (OxLDL) by macrophages and inhibits the proinflammatory properties of OxPL2-4. Here, to determine the role of OxPL in vivo in the context of atherogenesis, we generated transgenic mice in the Ldlr-/- background that expressed a single-chain variable fragment of E06 (E06-scFv) using the Apoe promoter. E06-scFv was secreted into the plasma from the liver and macrophages, and achieved sufficient plasma levels to inhibit in vivo macrophage uptake of OxLDL and to prevent OxPL-induced inflammatory signalling. Compared to Ldlr-/- mice, Ldlr -/- E06-scFv mice had 57-28% less atherosclerosis after 4, 7 and even 12 months of 1% high-cholesterol diet. Echocardiographic and histologic evaluation of the aortic valves demonstrated that E06-scFv ameliorated the development of aortic valve gradients and decreased aortic valve calcification. Both cholesterol accumulation and in vivo uptake of OxLDL were decreased in peritoneal macrophages, and both peritoneal and aortic macrophages had a decreased inflammatory phenotype. Serum amyloid A was decreased by 32%, indicating decreased systemic inflammation, and hepatic steatosis and inflammation were also decreased. Finally, the E06-scFv prolonged life as measured over 15 months. Because the E06-scFv lacks the functional effects of an intact antibody other than the ability to bind OxPL and inhibit OxLDL uptake in macrophages, these data support a major proatherogenic role of OxLDL and demonstrate that OxPL are proinflammatory and proatherogenic, which E06 counteracts in vivo. These studies suggest that therapies inactivating OxPL may be beneficial for reducing generalized inflammation, including the progression of atherosclerosis, aortic stenosis and hepatic steatosis.

B-1 cells are a unique subset of B cells that are positively selected for expressing autoreactive BCRs. We isolated RNA from peritoneal (B-1a, B-1b, B-2) and splenic (B-1a, marginal zone, follicular) B cells from C57BL/6 mice and used 5'-RACE to amplify the IgH V region using massively parallel sequencing. By analyzing 379,000 functional transcripts, we demonstrate that B-1a cells use a distinct and restricted repertoire. All B-1 cell subsets, especially peritoneal B-1a cells, had a high proportion of sequences without N additions, suggesting predominantly prenatal development. Their transcripts differed markedly and uniquely contained VH11 and VH12 genes, which were rearranged only with a restricted selection of D and J genes, unlike other V genes. Compared to peritoneal B-1a, the peritoneal B-1b repertoire was larger, had little overlap with B-1a, and most sequences contained N additions. Similarly, the splenic B-1a repertoire differed from peritoneal B-1a sequences, having more unique sequences and more frequent N additions, suggesting influx of B-1a cells into the spleen from nonperitoneal sites. Two CDR3s, previously described as Abs to bromelain-treated RBCs, comprised 43% of peritoneal B-1a sequences. We show that a single-chain variable fragment designed after the most prevalent B-1a sequence bound oxidation-specific epitopes such as the phosphocholine of oxidized phospholipids. In summary, we provide the IgH V region library of six murine B cell subsets, including, to our knowledge for the first time, a comparison between B-1a and B-1b cells, and we highlight qualities of B-1 cell Abs that indicate unique selection processes.

Liver X receptors (LXRs) are regulators of cholesterol metabolism that also modulate immune responses. Inactivation of LXR α and β in mice leads to autoimmunity; however, how the regulation of cholesterol metabolism contributes to autoimmunity is unclear. Here we found that cholesterol loading of CD11c⁺ cells triggered the development of autoimmunity, whereas preventing excess lipid accumulation by promoting cholesterol efflux was therapeutic. LXRβ-deficient mice crossed to the hyperlipidemic ApoE-deficient background or challenged with a high-cholesterol diet developed autoantibodies. Cholesterol accumulation in lymphoid organs promoted T cell priming and stimulated the production of the B cell growth factors Baff and April. Conversely, B cell expansion and the development of autoantibodies in ApoE/LXR-β-deficient mice was reversed by ApoA-I expression. These findings implicate cholesterol imbalance as a contributor to immune dysfunction and suggest that stimulating HDL-dependent reverse cholesterol transport could be beneficial in the setting of autoimmune disease.

Innate immunity utilizes evolutionarily conserved pattern recognition receptors (PRRs) to provide an early and effective response against Pathogen-Associated Molecular Patterns (PAMPs) on microbial pathogens and/or against Danger Associated Molecular Patterns (DAMPs) on endogenous modified-self structures. Atherosclerosis is a chronic inflammatory disease in which lipid peroxidation is greatly increased leading to the generation of OxLDL, which contains a variety of proinflammatory oxidation-specific neoepitopes (OSE), such as phosphocholine (PC) containing oxidized phospholipids (OxPL). Our group has shown that OSEs are DAMPs, to which has evolved a concerted innate immune response mediated by PRRs. For example, CD36, CRP and IgM E06 all recognize the PC of OxPL, but also the PC on apoptotic cells as well as the PC on the cell wall of S. pneumonia (but not as part of a lipid). Accordingly, we postulated that both endogenous DAMPs and exogenous PAMPs provide natural selection for PPR responses to PC. Malondialdehyde (MDA) is another prominent OSE target of three different PRR’s: SR-A, CFH, and the IgM NAb E014. Thus, we hypothesized that EO14 should also recognize an epitope/mimotope on an infectious pathogen. We screened a pathogen library with E014 and discovered it avidly bound to group A streptococcus (GAS). Because it was known that CFH also bound to GAS, and specifically to protein M, the major virulence factor of GAS, we used GAS with and without protein M to show that E014 specifically bound to protein M. Using a series of recombinant protein M fragments, we identified a 125 aa sequence required for binding. Using a synthetic peptide array to generate 15 aa-length overlapping peptides, we identified a 24 aa mimitope that E014 bound. We subsequently showed immunological cross reactivity between GAS, Protein M, the mimotope, and MDA in vitro and in vivo in mice and humans. Further, compared to immunization of mice with protein M, immunization with MDA-LDL provided partial protection against lethal infection with GAS. These data support the hypothesis that OSE are important targets of innate immunity and both oxidative events and pathogens have contributed to the natural selection of potent, shared innate immune responses to oxidation-specific epitopes.

Oxidized phospholipids (OxPL) are ubiquitously generated during inflammation, and found on apoptotic cells, OxLDL, and Lp(a). They facilitate uptake of OxLDL by macrophages (Mac) and mediate cellular inflammatory responses. The E06 natural IgM binds to the PC of OxPL, neutralizing biological effects and inhibiting OxLDL uptake by Mac. To determine the role of OxPL in atherogenesis, we generated transgenic mice expressing a single chain variant (scFv) of E06 in Ldlr background (E06-Tg). E06-scFv was secreted into plasma, bound to OxLDL and had sufficient titer to inhibit OxLDL uptake into Mac. E06-Tg or Ldlr mice were fed 1% Chol diet for 4, 7 or 12 months (n=12-15 mice/group). Plasma Chol was ~ 1600 mg/dL in all mice. Atherosclerosis decreased in E06-Tg mice: En face lesions decreased 57%, 34% and 28%, and aortic root lesions decreased 55%, 41% and 26% at 4, 7 and 12-months, respectively. OxLDL uptake by Macs was decreased: Thus, in E06-Tg mice, the uptake by peritoneal Mac of fluorescently-labeled OxLDL injected ip was decreased ~ 50%, as was peritoneal Mac Chol content. As Macs secrete E06-scFv, we performed BMT from E06-Tg donors into irradiated Ldlr recipients (n=10-12): This also decreased lesions 31% compared to BMT from control donors, even though plasma titers of E06-scFv were ~10% of Tg mice. Overexpression of E06-scFv was anti-inflammatory: Thus, in E06-Tg mice, both peritoneal and aortic wall resident macrophages exhibited decreased inflammatory gene expression, and phenotypic switches from M1 to M2 analyzed by RNAseq and FACS. Further, in E06-Tg mice, plasma SAA levels were reduced 32%, and hepatic steatosis was also decreased (-50% in both TG and Chol), as was hepatic inflammatory gene expression. Finally, E06-scFv attenuated both a progressive increase in aortic valve gradient (via echocardiography) and calcification in aged Ldlr mice. The E06-scFv lacks functional effects of an intact antibody other than the ability to “neutralize” OxPL. Thus, these data demonstrate that OxPL are profoundly proatherogenic and proinflammatory, which E06 counteracts in vivo . Neutralizing OxPL may therefore reduce the progression of atherosclerosis and cardiovascular events and more generally, represents a novel strategy to safely attenuate inflammation.

Adaptive immunity, which plays an important role in the development of atherosclerosis, is mediated by major histocompatibility complex (MHC)-dependent antigen presentation. In atherosclerotic lesions, macrophages constitute an important class of antigen presenting cells that activate adaptive immune responses to oxidized low-density lipoprotein (OxLDL). It has been reported that autophagy regulates adaptive immune responses by enhancing antigen presentation to MHC class II (MHC-II). In a previous study, we have demonstrated that SYK (spleen tyrosine kinase) regulates generation of reactive oxygen species (ROS) and activation of MAPK8/JNK1 in macrophages. Because ROS and MAPK8 are known to regulate autophagy, in this study we investigated the role of SYK in autophagy, MHC-II expression and adaptive immune response to OxLDL. We demonstrate that OxLDL induced autophagosome formation, MHC-II expression, and phosphorylation of SYK in macrophages. Gene knockout and pharmacological inhibitors of NOX2 and MAPK8 reduced OxLDL-induced autophagy. Using bone marrow-derived macrophages isolated from wild type and myeloid specific SYK knockout mice, we demonstrate that SYK regulates OxLDL-induced ROS generation, MAPK8 activation, BECN1-BCL2 dissociation, autophagosome formation and presentation of OxLDL-derived antigens to CD4(+) T cells. ldlr(-/-) syk(-/-) mice fed a high-fat diet produced lower levels of IgG to malondialdehyde (MDA)-LDL, malondialdehyde-acetaldehyde (MAA)-LDL, and OxLDL compared to ldlr(-/-) mice. These results provide new insights into the mechanisms by which SYK regulates MHC-II expression via autophagy in macrophages and may contribute to regulation of adaptive immune responses in atherosclerosis.