HMGB1–C1q complexes regulate macrophage functionby switching between leukotriene and specializedproresolving mediator biosynthesisTianye Liua,b, Alec Xianga, Travis Penga, Amanda C. Doranc,d,e, Kevin J. Traceyf, Betsy J. Barnesa, Ira Tabasc,d,e,Myoungsun Sona,b,1,2, and Betty Diamonda,b,1,2

aCenter for Autoimmune Musculoskeletal and Hematopoietic Diseases, The Feinstein Institute for Medical Research, Manhasset, NY 11030; bMD/PhDProgram at Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY 11549; cDepartment of Medicine, Columbia University,New York, NY 10032; dDepartment of Physiology, Columbia University, New York, NY 10032; eDepartment of Pathology and Cell Biology, ColumbiaUniversity, New York, NY 10032; and fThe Center for Biomedical Science, The Feinstein Institute for Medical Research, Manhasset, NY 11030

Edited by Lawrence Steinman, Stanford University School of Medicine, Stanford, CA, and approved September 9, 2019 (received for review May 2, 2019)

Macrophage polarization is critical to inflammation and resolutionof inflammation. We previously showed that high-mobility groupbox 1 (HMGB1) can engage receptor for advanced glycation endproduct (RAGE) to direct monocytes to a proinflammatory phenotypecharacterized by production of type 1 IFN and proinflammatorycytokines. In contrast, HMGB1 plus C1q form a tetramolecularcomplex cross-linking RAGE and LAIR-1 and directing monocytesto an antiinflammatory phenotype. Lipid mediators, as well ascytokines, help establish a milieu favoring either inflammationor resolution of inflammation. This study focuses on the inductionof lipid mediators by HMGB1 and HMGB1 plus C1q and theirregulation of IRF5, a transcription factor critical for the inductionand maintenance of proinflammatory macrophages. Here, we showthat HMGB1 induces leukotriene production through a RAGE-dependent pathway, while HMGB1 plus C1q induces specializedproresolving lipid mediators lipoxin A4, resolvin D1, and resolvinD2 through a RAGE- and LAIR-1–dependent pathway. Leukotrieneexposure contributes to induction of IRF5 in a positive-feedbackloop. In contrast, resolvins (at 20 nM) block IRF5 induction and pre-vent the differentiation of inflammatory macrophages. Finally, wehave generated a molecular mimic of HMGB1 plus C1q, which cross-links RAGE and LAIR-1 and polarizes monocytes to an antiinflamma-tory phenotype. These findings may provide a mechanism to controlnonresolving inflammation in many pathologic conditions.

leukotriene | SPMs | IRF5 | HMGB1 | C1q

Proinflammatory macrophages contribute to immune protec-tion in infection, but also to disease pathogenesis in auto-

immune diseases, atherosclerosis, Alzheimer’s disease, and manyconditions of chronic inflammation (1–4). Antiinflammatorymacrophages are important for cessation of inflammation andtissue repair (5). The degree and timing of proinflammatory andantiinflammatory macrophage induction are critical for maintain-ing immune homeostasis (6, 7). There is, therefore, a need tounderstand how to harness relevant pathways in order to regulateresolution of inflammation. However, the underlying mechanismsfor macrophage polarization are incompletely characterized.During inflammation, leukotrienes, produced from arachidonic

acid by 5-lipoxygenase (5-LO), help regulate leukocyte trafficking,chemotaxis, and diapedesis from the bloodstream into injuredtissue (8, 9). Specialized proresolving lipid mediators (SPMs),which include lipoxins, resolvins, protectins, and maresins, are alsoproduced from polyunsaturated fatty acid precursors (6, 7, 10, 11).These molecules block neutrophil migration and stimulatemacrophage uptake of cellular debris, which are processes re-quired for the resolution of inflammation (12). The generation ofleukotriene B4 (LTB4) (5S,6Z,8E,10E,12R,14Z-5,12-dihydroxy-6,8,10,14-icosatetraenoic acid) requires the function of 5-LO.When 5-LO is phosphorylated, it translocates to the nuclearmembrane and promotes leukotriene production (13, 14). In

contrast, when 5-LO is not phosphorylated, it localizes in thecytoplasm and promotes SPM production (15). Resolvins, es-pecially resolvin D1 (RvD1) (7S,8R,17S-trihydroxy-docosa-4Z,9E,11E,13Z,15E,19Z-hexaenoic acid), contribute to resolu-tion of inflammation by promoting nuclear exclusion of 5-LOand, in a positive-feedback loop, by enhancing production ofthese SPMs. Other SPMs such as protectins or maresins do notdirectly require 5-LO activation (15, 16). The antiinflammatoryeffects of lipoxin A4 (LXA4) (5S,6R,15S-trihydroxy-eicosa-7E,9E,11Z,13E-tetraenoic acid), RvD1, RvD2 (7S,16R,17S-trihydroxy-docosa-4Z,8E,10Z,12E,14E,19Z-hexaenoic acid), andtheir ability to reduce production of inflammatory cytokinessuch as IFN-γ, TNFα, and IL-1β are well established (17–19).C1q is a modulator of inflammation and repair that can main-

tain monocyte quiescence or cooperate with damage-associatedmolecular patterns (DAMPs) to induce antiinflammatory macro-phages (20, 21). C1q is composed of globular heads and a collagen-like tail that binds to the leukocyte-associated Ig-like receptor-1(LAIR-1), a transmembrane protein of the Ig superfamily (22–24).High-mobility group box 1 (HMGB1) is a DAMP that is elevatedin the serum of SLE patients (25–28). Extracellular HMGB1 bindsnucleic acid cargo, engages the receptor for advanced glycation

Significance

Monocyte to macrophage differentiation is critical to inflamma-tion and resolution of inflammation and can be regulated byhigh-mobility group box 1 (HMGB1) and HMGB1 plus C1q, re-spectively. These are 2 evolutionarily old and highly conservedmolecules. While HMGB1 causes a positive-feedback loop be-tween the proinflammatory lipid mediator leukotriene B4 (LTB4)and IRF5, an important regulator of inflammatory macrophagepolarization, HMGB1 plus C1q increases production of specializedproresolving lipid mediators (SPMs) lipoxin A4, resolvin D1, andresolvin D2, which prevent IRF5 transcription. As nonresolvinginflammation is a common condition, we designed an HMGB1–C1q mimetic peptide that can polarize monocytes to an antiin-flammatory phenotype in vitro and in vivo.

Author contributions: T.L., I.T., M.S., and B.D. designed research; A.X., T.P., and A.C.D.performed research; T.L., I.T., M.S., and B.D. analyzed data; and T.L., K.J.T., B.J.B., I.T., M.S.,and B.D. wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1M.S. and B.D. contributed equally to this work.2To whom correspondence may be addressed. Email: mson@northwell.edu or bdiamond@northwell.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1907490116/-/DCSupplemental.

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end product (RAGE), and transports nucleic acid to endosomalToll-like receptors (TLRs) 7 and 9 (27, 29). It is known thatHMGB1-induced macrophage polarization of monocytes de-pends on its interaction with 5-LO and LTB4 receptor BLT1pathways (30).The transcription factor IFN regulatory factor 5 (IRF5) has

been identified as a regulator of proinflammatory macrophagepolarization (31–33). IRF5 directly induces transcription ofproinflammatory cytokines and type I IFN while repressingtranscription of IL-10 and TGFβ in macrophages (32, 34).Polymorphisms of the IRF5 gene leading to high expression inmonocytes are associated with rheumatoid arthritis and sys-temic lupus erythematosus (SLE) (35). The relationship be-tween IRF5 expression and leukotriene or resolvin productionhas not been fully explored. Here, we further explore this re-lationship. We also address the involvement of C1q and otherSPMs in leukotriene production and macrophage polarization.

ResultsHMGB1 Leads to LTB4 Production and HMGB1 Plus C1q Leads to SPMProduction in Human Monocytes. We previously showed thatHMGB1 induces the production of proinflammatory cytokines byhuman monocytes in a RAGE-dependent pathway (20). We,therefore, asked whether leukotriene production is also a com-ponent of the proinflammatory phenotype of HMGB1-stimulatedmonocytes. We assessed LTB4 in culture supernatant of HMGB1-stimulated monocytes. LTB4 production was significantly inducedby HMGB1 at 1 h. While it decreased at 3 h, it was still signifi-cantly higher than in supernatant of unstimulated cells (Fig. 1A).Just as HMGB1 plus C1q suppresses induction of proinflammatorycytokines and induces, instead, the production of IL-10, monocytes

stimulated with HMGB1 plus C1q secreted significantly less LTB4than monocytes stimulated with HMGB1 alone. They insteadproduced SPMs such as LXA4, RvD2, and RvD1 (Fig. 1 B–D).C1q alone did not lead to the induction of SPMs. Levels of SPMswere increased at 1 to 3 h and were maintained for up to 6 h.

HMGB1 and C1q Reciprocally Regulate Phosphorylation and NuclearLocalization of 5-LO. LTB4 is generated from arachidonic acid bynuclear 5-LO, and lipoxins are produced from arachidonic acid bycytosolic 5-LO (15). We, therefore, evaluated 5-LO localization inHMGB1 and HMGB1-plus-C1q–treated monocytes and de-termined the ratio of nuclear to cytosolic 5-LO. 5-LO was localizedin perinuclear regions in HMGB1-exposed cells while it was lo-calized in the cytosol of HMGB1-plus-C1q–exposed cells (Fig. 2A).Additionally, serine 271 phosphorylation of 5-LO, which promotesits nuclear localization, was induced at 1 h by HMGB1 but not byHMGB1 plus C1q (Fig. 2B). This suggests that HMGB1 plus C1qprevents HMGB1-induced LTB4 synthesis by inhibiting phos-phorylation and nuclear localization of 5-LO.In order to understand whether the RAGE and LAIR-1

pathways contribute to HMGB1 induction of LTB4, we trans-fected human monocytes with RAGE-specific or LAIR-1–specificsmall interfering RNA (siRNA). LTB4 production induced byHMGB1 was significantly decreased in monocytes transfectedRAGE-siRNA compared to control siRNA-transfected monocytesbut not in LAIR-1–knockdown monocytes, confirming a requirementof RAGE in HMGB1-stimulated LTB4 production (Fig. 3A).RvD2 production upon HMGB1-plus-C1q stimulation was di-minished in both RAGE-knockdown monocytes and in LAIR-1–knockdown monocytes, respectively, showing that both RAGEand LAIR-1 are required for HMGB1-plus-C1q–induced RvD2

Fig. 1. HMGB1 induces LTB4, and HMGB1 plus C1q induces SPMs in human monocytes. Induction of (A) LTB4, (B) LXA4, (C) RvD2, and (D) RvD1 by HMGB1 and/orC1q. Human monocytes were treated with HMGB1 (1 μg/mL) and/or C1q (25 μg/mL) in X-Vivo 15 serum-free medium. LTB4, LXA4, RvD2 (after 1, 3, and 6 h), andRvD1 (after 3 h) in supernatant were measured by ELISA (mean ± SD of triplicates; n = 3 for 1 and 6 h; n = 5 for 3 h). ***P < 0.001; ns, not significant (1-way ANOVA).

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production (Fig. 3A). We confirmed a requirement for RAGE inLTB4 production (Fig. 3B) and a requirement for RAGE andLAIR-1 in RvD2 production in murine monocytes (Fig. 3C).SH2 domain-containing protein tyrosine phosphatase-1 (SHP-1)is a tyrosine phosphatase that binds phosphorylated LAIR-1 (36).To understand whether SHP-1 is critical to the production ofproresolving lipid mediators, we used siRNA to diminish SHP-1 expression. HMGB1-plus-C1q–induced production of RvD2 inhuman monocytes was decreased in SHP-1 siRNA-transfectedmonocytes (Fig. 3D).

IRF5 Is Increased in HMGB1-Stimulated Macrophages and PositivelyRegulates LTB4 Production. To determine whether HMGB1-stimulated macrophages exhibit high expression of IRF5, as hasbeen shown in proinflammatory macrophages triggered by en-gagement of other surface receptors, we assessed the mRNA andprotein levels of IRF5 4 h after HMGB1 stimulation. IRF5 mRNAand protein expression were increased in HMGB1-stimulatedmacrophages (Fig. 3E). In contrast, HMGB1 plus C1q did notinduce IRF5. HMGB1-mediated IRF5 expression did not occur inhuman monocytes transfected with RAGE siRNA, demonstratingthat IRF5 expression is downstream of RAGE engagement (Fig.3F). TNFα up-regulation was also blocked in monocytes trans-fected with RAGE siRNA (SI Appendix, Fig. S1A), confirmingresults of our previous studies, which showed that TNFα inductionby HMGB1 was mediated through RAGE (33). We confirmed inRAGE-deficient and wild-type (WT) mouse monocytes thatRAGE is necessary for induction of IRF5 by HMGB1 (Fig. 3G).We next examined whether IRF5 modulates LTB4 production

in monocytes. We observed that IRF5-specific siRNA abrogated

HMGB1-induced LTB4 secretion (Fig. 3H). RvD2 production inHMGB1-plus-C1q–triggered cells was not affected by IRF5siRNA (SI Appendix, Fig. S1B). We then confirmed IRF5-dependent LTB4 secretion in mouse monocytes deficient inIRF5. HMGB1-induced LTB4 production was significantlyless in IRF5-deficient monocytes than in WT monocytes (Fig.3I). Reciprocally, when IRF5 was overexpressed in humanmonocytes, the basal production of LTB4 was increased,suggesting a role for IRF5 upstream of LTB4 (SI Appendix,Fig. S1C). These results suggest that the IRF5 pathway is thecritical mediator of LTB4 production.

The Leukotriene Pathway Modulates IRF5 Transcription. To determinewhether HMGB1-induced IRF5 expression is mediated by path-ways downstream of LTB4, we used U75302, an LTB4 receptorBLT1 inhibitor and LY255283, an LTB4 receptor BLT2 inhibi-tor to block leukotriene signaling (30, 37, 38). Blockade of BLT1and BLT2, both individually and together led to a decrease inHMGB1-induced IRF5 transcription with the combination of in-hibitors having the strongest effect (Fig. 4A). To confirm the ob-servation, we showed that HMGB1-induced IRF5 transcriptionwas abolished when either or both receptors were diminished bysiRNA (Fig. 4B). We next asked whether LTB4 can induceIRF5 transcription. IRF5 transcription increased under LTB4stimulation. BLT1 and BLT2 inhibitors, both individually and to-gether, decreased LTB4-induced IRF5 transcription. (Fig. 4C).Reciprocally, RvD1 and RvD2 (20 nM) and LXA4 (20 nM)suppressed induction of IRF5 by HMGB1 (Fig. 4D). Thus, SPMsproduced by 5-LO can prevent expression of IRF5 inducedby HMGB1 stimulation.

HMGB1-Induced Peritonitis. Having established pathways ofleukotriene and SPM production in primary human monocytes, wenext sought to explore mechanisms underlying proinflammatoryand antiinflammatory macrophage polarization and the contributionof lipid mediators in an in vivo mouse model. We determined thatHMGB1, injected intraperitoneally (i.p.) into C57BL/6 mice, couldinduce an inflammatory peritonitis. When HMGB1 (10 μg/mouse)was injected i.p. into mice, the peritoneal exudate was character-ized initially by increased levels of LTB4 followed by increasingRvD2. (Fig. 5A). In contrast, when mice were given HMGB1plus C1q i.p., there was more RvD2 in the peritoneal exudateand a reduction in LTB4. We also observed that the level ofTNFα was high in HMGB1-induced exudates, while IL-10 washigh in HMGB1-plus-C1q–induced exudates (SI Appendix, Fig.S2). These in vivo results are consistent with the data obtainedwith human monocytes in which proinflammatory macrophagesmaking leukotriene are generated by exposure to HMGB1 andantiinflammatory macrophages making SPMs by exposure toHMGB1 plus C1q. Six hours after HMGB1 or HMGB1-plus-C1qinjection, macrophages (CD11b+Ly6G−) were isolated from peri-toneal exudates. Macrophages from mice injected with HMGB1exhibited a high level of TNFα and IRF5 mRNA, whereas mac-rophages from mice injected with HMGB1 plus C1q exhibited ahigher level of Mer, Arg1, and TGFβ mRNA, which are anti-inflammatory markers in murine macrophages (Fig. 5B). Moreover,a higher frequency of Merhigh macrophages was observed in miceinjected with HMGB1 plus C1q than in mice injected withHMGB1 (Fig. 5C). Merhigh and Merlow macrophages were isolatedand cultured ex vivo to confirm their proinflammatory or anti-inflammatory phenotype. Merlow cells produced more LTB4 thanMerhigh cells, whereas Merhigh cells secreted more RvD2 thanMerlow cells (Fig. 5D). We also examined the effect of LTB4 re-ceptor antagonism in this peritonitis model. CD11b+Ly6G− cellsfrom peritoneal exudates of mice pretreated with LTB4 receptorantagonists exhibited decreased TNFα and IRF5 mRNA and in-creased Mer and Arg1 mRNA compared to untreated controlsupon HMGB1 stimulation (Fig. 5E).

Fig. 2. HMGB1 and C1q reciprocally regulate phosphorylation and nuclearlocalization of 5-LO. (A) Nuclear localization of 5-LO induced by HMGB1 and/orC1q stimulation for 1 h; nucleus marked by PI (blue) and 5-LO stained withrabbit anti–5-LO and Alexa Fluor 488 anti-rabbit IgG (red). The ratio of nuclear/nonnuclear 5-LO was determined in more than 80 cells in each condition(mean ± SEM; n = 4). (Scale bar, 10 μm.) *P < 0.05 and **P < 0.01 (1-wayANOVA). (B) Immunoblot for serine-271 phosphorylation of 5-LO in total celllysates from HMGB1 or HMGB1-plus-C1q–stimulated monocytes (1 h). The foldinduction of phospho-5-LO in HMGB1 and/or C1q-stimulated monocytes iscompared to untreated samples from 3 independent experiments. *P <0.05 and **P < 0.01 (1-way ANOVA).

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Fig. 3. HMGB1 induces LTB4 through RAGE and IRF5, while HMGB1 plus C1q induces RvD2 through RAGE and LAIR-1. (A) LTB4 production byHMGB1 stimulation and RvD2 production by HMGB1-plus-C1q stimulation in RAGE or LAIR-1 siRNA-transfected human monocytes. Twenty-four hours aftertransfection with siRNA, cells were stimulated with HMGB1 alone (1 μg/mL), C1q alone (25 μg/mL), or HMGB1 plus C1q. HMGB1-induced LTB4 (after 1 h) andHMGB1-C1q–induced RvD2 (after 3 h) were measured in culture supernatant by ELISA. Knockdown efficiencies of RAGE and LAIR-1 were assessed by qRT-PCR.RE, relative expression. *P < 0.05 and ***P < 0.001 (1-way ANOVA or t test). n = 4. (B and C) LTB4 production by HMGB1 in RAGE-deficient monocytes andRvD2 production by HMGB1 plus C1q in RAGE- or LAIR-1–deficient monocytes. Splenic monocytes from WT mice or RAGE-deficient monocytes were stim-ulated with HMGB1 for 1 h or HMGB1 plus C1q for 3 h. LTB4 and RvD2 measured by ELISA. *P < 0.05, **P < 0.01, and ***P < 0.001 (1-way ANOVA or t test). (D)Reduced RvD2 production by HMGB1-plus-C1q stimulation in SHP-1 siRNA-transfected human monocytes. Knockdown efficiency of SHP-1 were determined byqRT-PCR. n = 3. (E) Total cell lysates were subjected to Western blot with antibodies specific for IRF5 and β-actin. Numbers indicate the relative signal intensitycompared to actin. Bar graphs show fold change compared to the intensity of IRF5 in untreated samples from 3 independent experiments. (F) RAGE-specificsiRNA-transfected human monocytes showed significantly decreased IRF5 levels following HMGB1 stimulation for 4 h. IRF5 relative expression was de-termined by qRT-PCR. n = 4. (G) Splenic monocytes from WT mice or RAGE-deficient mice were isolated and stimulated with HMGB1 and/or C1q for 4 h. Eachdot represents an individual animal. (H) IRF5-specific siRNA abrogates HMGB1-stimulated LTB4 production 1 h after HMGB1 stimulation, n = 4. Knockdownefficiency of IRF5 was determined by qRT-PCR. (I) IRF5-deficient monocytes showed reduced LTB4 secretion following HMGB1 stimulation. Splenic monocytesfrom WT mice or IRF5-deficient mice were isolated and stimulated with HMGB1 and/or C1q for 1 h. Each dot represents an individual animal (mean ± SEM of5 mice/group). *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant (1-way ANOVA).

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An HMGB1-Plus-C1q Mimetic Cross-Links RAGE and LAIR-1 and InducesAntiinflammatory Phenotypes.Having shown that HMGB1 inducesLTB4 in monocytes in a RAGE-dependent pathway, and that thisprocess, in turn, augments IRF5 transcription and a proinflammatorypolarization of macrophages, and that HMGB1 plus C1q inducesRvD2 in a RAGE- and LAIR-1–dependent pathway leading tosuppression of IRF5 transcription and a proresolving polarizationof macrophages both in vitro and in vivo, we wanted to develop amolecule that could harness these pathways therapeutically. We,therefore, generated a peptide containing a RAGE-binding regionof HMGB1 (B-box) (39), a short linker sequence (40), and a C1qpeptide that binds LAIR-1 and induces its phosphorylation (Fig. 6A and B). This peptide was able to cross-link RAGE and LAIR-1in a proximity ligation assay (Fig. 6C) and hence is termedRAGE–LAIR-1 cross-linking peptide (RLCP). Human monocytes

incubated with HMGB1 plus RLCP, similar to HMGB1 plus C1q,exhibited decreased proinflammatory polarization and decreasedIRF5 mRNA compared to monocytes incubated with HMGB1alone (Fig. 6D). RLCP in the presence of HMGB1 and by itselfinduced IL-10 transcription in human monocytes in vitro (Fig. 6E).WT mice injected with HMGB1 plus RLCP together producedless LTB4, less prostaglandin E2 (PGE2), and more RvD2 in theirperitoneal exudates than those injected with HMGB1 alone after3 h. (Fig. 6 F–H). Mice injected with RLCP had more alternativelyactivated macrophages (CD206+CD11b+Ly6G−) in the peritonealexudate than mice injected with HMGB1 (Fig. 6I). Thus, RLCPfunctions similarly to HMGB1 plus C1q in both human monocytesand in the mouse peritonitis model to induce antiinflammatorymacrophage polarization.

Fig. 4. LTB4 and RvD2 pathways reciprocally regulate IRF5 gene expression. (A) Blockade of the LTB4 pathway diminished HMGB1-induced IRF5 transcription. Humanmonocytes were preincubated with LTB4 receptor BLT1 antagonist (LY255283; 200 nM), LTB4 receptor BLT2 antagonist (U75304; 200 nM), or both for 15 min followedby HMGB1 for 4 h at 37 °C. IRF5 mRNA was assessed by qRT-PCR. n = 3. (B) Human monocytes transfected with siRNA for BLT1, BLT2, or both were treated withHMGB1 after 24 h for 4 h. Relative expression of IRF5 and knockdown efficiencies were determined by qRT-PCR. n = 3. (C) Humanmonocytes were preincubated withLTB4 receptor BLT1 antagonist (LY255283; 200 nM) and/or LTB4 receptor BLT2 antagonist (U75304; 200 nM) for 15 min followed by LTB4 (200 nM) for 4 h. IRF5 mRNAassessed by qRT-PCR. (D) RvD1, RvD2, LXA4, and LXA4 analog suppress IRF5 mRNA. Human monocytes were preincubated with RvD1 (20 nM), RvD2 (20 nM), or LXA4

(20 nM) for 15 min followed by HMGB1 for 4 h. IRF5 mRNA induced by HMGB1 was assessed by qRT-PCR. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001 (1-way ANOVA).

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Fig. 5. C1q promotes antiinflammatory macrophages in HMGB1-induced peritonitis. C57BL/6 WT mice were given HMGB1 i.p. (10 μg/mouse) with or without C1q(200 μg/mouse). (A) Peritoneal exudates frommice given HMGB1 and/or C1qwere obtained at 3, 6, and 12 h, and LTB4 and RvD2weremeasured by ELISA. Each dotrepresents an individual animal. (B) Six hours after injection with HMGB1 or HMGB1 plus C1q, CD11b+Ly6G− cells were isolated from peritoneal exudates andanalyzed for TNFα, IRF5, Mer, Arg1, and TGFβ mRNA. n = 3. *P < 0.05, **P < 0.01, and ***P < 0.001 (t test). (C) The percentage of Mer-expressing cells inCD11b+Ly6G− cells was higher in mice injected with HMGB1 plus C1q than in mice injected with HMGB1 alone. n = 3. (D) Isolated Merhigh and Merlow cells wereseparately cultured in X-Vivo medium. LTB4 (1 h) and RvD2 (3 h) were measured in the culture supernatant. n = 3. (E) WT mice were injected with HMGB1 i.p.(10 μg/mouse) or HMGB1-plus-BLT1 inhibitor (LY255283; 10 mg/kg). Peritoneal exudate cells were isolated, and proinflammatory and antiinflammatory markers wereassessed on CD11b+Ly6G− cells by qRT-PCR (3 h). n = 3. *P < 0.05 (t test).

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Fig. 6. RAGE and LAIR-1 cross-linking peptide, RLCP, induces RvD2 and abolishes LTB4 induction by HMGB1. (A) Amino acid sequence of a fusion proteincontaining B box, a RAGE binding region of HMGB1 and a C1q tail peptide linked by a flexible (Gly4Ser)3 linker (RAGE and LAIR-1 cross-linking peptide [RLCP]). (B)RLCP phosphorylates LAIR-1 in a phospho-immunoreceptor array. RLCP (500 nM) was incubated with fresh human monocytes for 15 min at 37 °C. Relativequantification for the phosphorylation of LAIR-1 was normalized to control spots. n = 3. (C) Proximity ligation assay performed on human monocytes showed thatRLCP (500 nM, 15 min) cross-links RAGE and LAIR-1. Red fluorescent dots represent proximity between RAGE and LAIR-1; blue represents nuclear staining withDAPI. Original magnification≥ 40×. One of 3 representative experiments is shown. (Scale bar, 10 μm.) (D and E) Human monocytes were incubated for 4 h withHMGB1 (1 μg/mL) and/or C1q (25 μg/mL), HMGB1 plus RLCP (500 nM), or RLCP alone (500 nM). IRF5 and IL-10 mRNA expression were assessed by qRT-PCR. n = 3.(F–H) C57BL/6 WT mice were given HMGB1 i.p. (10 μg/mouse) and/or C1q (200 μg/mouse), HMGB1 plus RLCP (500 μg/mouse), or RLCP (500 μg/mouse). Peritonealexudates were isolated, and LTB4 and PGE2 were measured by ELISA 3 h after injection. Each dot represents an individual animal (mean ± SEM). RvD2 wasmeasured by ELISA 6 h after injection. (I) Percentage of CD206+ cells in CD11b+Ly6G− mouse peritoneal exudates from HMGB1 i.p. (10 μg/mouse),HMGB1 plus C1q (200 μg/mouse), or HMGB1 plus RLCP (500 μg/mouse). Each dot represents an individual animal. *P < 0.05; **P < 0.01; ***P < 0.001;ns, not significant (1-way ANOVA).

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Together, these observations describe a positive-feedback loopin inflammatory macrophages (IRF5 and LTB4) and an inhibitorypathway initiated by C1q (Fig. 7), suggesting that a balanced levelof HMGB1 and C1q is critical in immune homeostasis.

DiscussionThe mammalian immune system relies on a fine balance of manyinterrelated stimulatory and inhibitory pathways. Chronic in-flammatory conditions are caused by disruptions of this delicatehomeostasis, leading to more active stimulatory pathways and thefailure of inflammation resolution. Lipid mediators, such as leu-kotrienes and SPMs, help maintain immune homeostasis by pro-moting inflammation and resolution, respectively. Upsetting theirbalance leads to changes in local tissue microenvironments thatalter the timing and duration of inflammation and has been linkedto various chronic inflammatory conditions (41). For example,SLE is a disease of nonresolving inflammation, characterized bythe presence of autoantibodies and systemic inflammation. Thehigh percent (80 to 90%) of C1q-deficient individuals develop SLEclearly shows that C1q helps maintain immune homeostasis (24,42). Interestingly, there is clinical evidence that derangements inlipid mediators are associated with SLE disease activity. Elevationsof urine leukotriene levels are seen in patients with active SLE(43), and LXA4, an SPM, has been proposed as a biomarker topredict prognosis and response to therapy (44).We previously described that relative levels of HMGB1 and C1q

reciprocally regulate proinflammatory and proresolving cytokinesin RAGE- and LAIR-1–dependent pathways (20). Here, we fur-ther demonstrate that HMGB1 and C1q utilize the same pathwayto regulate LTB4 and SPM production through regulation ofIRF5. These observations both expand on the existing knowledgeof lipid mediator pathways and further explore the mechanisms bywhich HMGB1 and C1q coregulate immune homeostasis, and howthe absence of C1q promotes SLE.LTB4 and its activator 5-LO contribute to inflammation through

increased vascular permeability, attraction, and activation of leu-kocytes (45, 46). It is known that phosphorylation status and cel-lular localization of 5-LO determine its activity and affect its rolein LTB4 versus SPM production (15). Our finding that HMGB1and HMGB1-plus-C1q stimulations result in distinct patterns of

5-LO cellular localization and phosphorylation serves to explaintheir observed effects on LTB4 production. We demonstratedthat HMGB1 stimulation of monocytes can lead to an increasein LTB4 production in a RAGE-dependent manner. It is knownthat RAGE-mediated vascular smooth muscle cell proliferation isenhanced by HMGB1 through 5-LO (47). Perinuclear localizationof 5-LO is involved not only in leukotriene production but also inthe activation of NF-κB in granulocytes (48). 5-LO forms a mo-lecular complex with NF-κB and 5-LO inhibitors inhibit NF-κBactivation (49). In the MRL/lpr mouse model of SLE, enhancedrenal leukotriene production is associated with increased severityof lupus nephritis, whereas a leukotriene receptor antagonist im-proves renal pathology (50).SPMs are a family of lipids that actively promote resolution

and tissue repair without compromising host defenses. Inductionof SPMs has been recognized to have therapeutic potential (51).SLE patients have lower serum levels of RvD1, suggesting a role ofSPM dysregulation in the disease (52). We show that RvD1,RvD2, and LXA4 productions are all downstream of HMGB1-plus-C1q stimulation in a RAGE- and LAIR-1–dependent man-ner. These findings suggest that SPM production is part of theantiinflammatory polarization of monocytes previously describedin HMGB1–C1q cross-linking of RAGE and LAIR-1. It is alsoknown that LXA4, RvD1, and RvD2 can activate an M1-to-M2macrophage phenotype switch in vivo in adipocyte tissue in anautocrine or paracrine fashion through engagement of G-protein–coupled receptors or formyl peptide receptor 2 (ALX/FPR2) (53).HMGB1-plus-C1q–induced SPM production appears to occur in asimilar fashion and can contribute to sustaining the antiinflamma-tory phenotype of monocyte/macrophages.IRF5 is a member of the IFN regulatory factor family of tran-

scription factors that modulate inflammatory immune responses innumerous cell types (54). It is well established that IRF5 plays animportant role in various inflammatory conditions. In atheroscle-rosis, IRF5 maintains proinflammatory macrophages within ath-erosclerotic lesions, impairs efferocytosis, and promotes lesiongrowth (55). Pattern recognition receptor (PRR)-stimulated M1macrophages require IRF5 to enhance glycolysis, which is char-acteristic of M1 polarization and is a central mediator of inflam-mation (56). The role of IRF5 in SLE pathogenesis is well

Fig. 7. Schematic of the IRF5 and LTB4 pathway. HMGB1 engages RAGE and induces both LTB4 production and IRF5 expression. LTB4 binds its receptor to stimulatefurther induction of IRF5 in a positive-feedback loop, resulting in the additional production of LTB4 and proinflammatory macrophage polarization. HMGB1 plusC1q induces production of specialized proresolving mediators (lipoxin A4, resolvin D1, and resolvin D2), which function to inhibit IRF5 expression through theirrespective receptors. HMGB1-C1q mimetic (RAGE and LAIR-1 cross-linking peptide [RLCP]) can polarize monocytes to an antiinflammatory phenotype.

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established. IRF5 leads to production of type I IFN in myeloid cells(57). It was shown that IRF5 deficiency ameliorates SLE in mousemodels (58–60). Moreover, there are IRF5 polymorphisms thatassociate with high IRF5 in monocytes and SLE (56, 61). Wedemonstrated a positive-feedback loop between IRF5 and SPMs.LTB4 production depends on IRF5, and that disruption of LTB4production, by antagonizing or knocking down the LTB4 receptorsBLT1/2, leads to a decrease in IRF5 expression. In contrast, ex-posure to nanomolar concentrations of SPMs such as RvD1, RvD2,and LXA4, decreased HMGB1-stimulated IRF5 expression (Fig.7). These are far more potent than the current drugs used in thetreatment of SLE and other diseases associated with uncontrolledinflammation (62). RvD1 is an SPM known to limit 5-LO peri-nuclear localization and LTB4 synthesis through a calcium-activatedkinase-dependent pathway (15). Given that IRF5 and LTB4 arecodependent and mutually enhancing, it is reasonable to speculatethat the observed decrease in LTB4 following HMGB1-plus-C1qstimulation can be attributed, at least in part, to an inhibitory effectof SPMs produced by the HMGB1-plus-C1q pathway on 5-LOnuclear localization and phosphorylation, leading to a disrup-tion of the positive-feedback loop between IRF5 with LTB4.Although we showed that LTB4 receptor antagonists and

resolvins can all decrease HMGB1-induced IRF5 expression, we didnot see the same effect in LPS-stimulated monocytes. Thus, thepositive-feedback loop between LTB4 and IRF5 does not developwhen IRF5 is induced by TLR4 activation (SI Appendix, Fig. S3). Ofnote, the LTB4 receptor, which can associate with RAGE, has notbeen shown to associate with TLR4 (63). Fully delineating the in-terrelated pathways involving IRF5 and LTB4 in HMGB1-stimulatedmonocytes will require further study. Nonetheless, the data presentedhere provide a mechanism for the coordination of inflammation, withproduction of both inflammatory cytokines and inflammatory lipidmediators and the coordination of resolution of inflammation, withproduction of both IL-10 and proresolving lipid mediators.The RAGE and LAIR-1 cross-linking peptide that mimics the

effect of HMGB1 plus C1q not only further confirmed thedownstream effects of RAGE-LAIR-1 cross-linking identified inour study, it also demonstrated that this pathway can be harnessedfor therapeutic potential. We showed that RLCP was able to in-duce proresolving macrophage differentiation, increase SPM pro-duction, and decrease LTB4 production and IRF5 expression bothin vitro and in vivo. RLCP induces SPMs and also blocks IRF5induction. It is important to note that human monocytes use theenzyme eicosanoid oxidoreductase to rapidly inactivate LXA4,RvD1, and RvD2 (64–66). The amount of these SPMs inducedin vitro were measured in supernatants; therefore, total cellularproduction of these lipid mediators may be underestimated.The 5-LO inhibitor, zileuton, is approved for treating asthma; onestudy suggested that it may be effective in managing SLE (67, 68).Our identification of RAGE and HMGB1 as upstream compo-nents of 5-LO nuclear translocation and the evidence thatHMGB1 plus C1q induce SPMs that suppress leukotriene pro-duction open up opportunities for the designs of therapeutics thatregulate lipid abnormalities in SLE. Together with its observeddown-regulation of IRF5 and macrophage polarization, RLCP ora functionally similar molecule may address multiple aspects of

SLE pathogenesis or other diseases of chronic inflammation.Although there are limitations in extrapolating from our short-term assays to chronic conditions, we showed in our previous studythat these proximal events involving HMGB1 and C1q can haveimpact in adaptive immunity. HMGB1-plus-C1q stimulation sup-pressed monocyte-to-dendritic cell differentiation and decreasedthe ability of macrophages to stimulate T cells (20). Continuedinvestigation of the immunomodulatory effects of HMGB1 andHMGB1 plus C1q can greatly enhance our understanding of SLEand provide approaches to SLE therapeutics. These findings mayalso illuminate mechanisms in cancer immunology. It is known thatC1q is overexpressed in the stroma and vascular endothelium ofmany malignant tumors and serves as a cancer-promoting factor intumor microenvironments (69). LAIR-1 is also known to beoverexpressed in several human tumors (70, 71). It is reasonable tospeculate that C1q and LAIR-1 may interact in this context topromote tumor escape from immune control, and that the path-ways we have explored are involved in immune function, in general.

Materials and MethodsMice and Study Approval. C57BL/6J WT mice, Lysozyme2-cre mice, and LAIR-1–deficient C57BL/6 were purchased from The Jackson Laboratory. Lysozyme2-cremice and LAIR-1–deficient C57BL/6 were bred in our facility to generate mye-loid cell-specific LAIR-1–deficient mice (LAIR-1 cKO). RAGE-deficient mice andIRF5-deficient mice were maintained in our facility (72, 73). This study wascarried out in strict accordance with recommendations in Guide for the Careand Use of Laboratory Animals of the NIH (74). The protocol was approved bythe Institutional Animal Care and Use Committee of the Feinstein Institute forMedical Research. Human study approval: This study is IRB exempt.

Monocyte Isolation and Cell Culture. Human peripheral blood mononuclearcells were isolated from whole human blood by density gradient centrifugationusing Ficoll-Paque Plus (GE Healthcare). Blood was obtained from healthy do-nors (New York Blood Center). Human approval: This study is IRB exempt.Monocytes were enriched by negative selection using a human monocyte en-richment kit (Stem Cell Technology). For monocytes from mice, cells wereenriched by positive selection kit (Stem Cell Technology). The purity of mono-cytes (≥90%) was determined by flow cytometer (LSRII; BD Biosciences). Purifiedmonocytes (2–5 × 106 cells per mL) were cultured in U-bottom 96-well plates inX-Vivo 15 serum-free medium (Lonza) with or without HMGB1 (1 μg/mL), C1q(25 μg/mL), and RLCP (500 nM) treatment and harvested at the indicated times.

Lipid Mediator Analysis. Culture supernatant or mouse peritoneal lavage wascollected, and LTB4, RvD1, RvD2, and PGE2 were quantitated by ELISA (CaymanChemical) following the manufacturer’s instruction. LXA4 was quantitatedsimilarly (Oxford Biomedical Research).

Statistics. Statistical analyses were performed using Prism 7 (GraphPad), andvalues of P < 0.05 were considered significant. Student’s t test analyzed thevariance of mean values between 2 groups for unpaired observations. Groupdifferences were tested with 1-way ANOVA followed by Tukey’s correctionfor multiple comparisons.

ACKNOWLEDGMENTS. We thank Heriberto Borrero, Christopher Colon,Amanda Chan, and Bruce T. Volpe for discussion and technical assistance.This work was supported by grants from the NIH/National Institute ofArthritis and Musculoskeletal and Skin Diseases (K01AR065506 [M.S.]), theNIH/National Institute of Allergy and Infectious Diseases (R01AI135063 [M.S.]and P01AI102852 [B.D.]), and the NIH (R01HL127464 and R35HL145228I [I.T.]).

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