Contribution of Adipose-Derived Factor D/Adipsin to Complement

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Contribution of Adipose-Derived Factor D/Adipsin to Complement Alternative Pathway Activation: Lessons from Lipodystrophy This information is current as of September 25, 2021. Xiaobo Wu, Irina Hutson, Antonina M. Akk, Smita Mascharak, Christine T. N. Pham, Dennis E. Hourcade, Rebecca Brown, John P. Atkinson and Charles A. Harris J Immunol 2018; 200:2786-2797; Prepublished online 12 March 2018; Downloaded from doi: 10.4049/jimmunol.1701668 http://www.jimmunol.org/content/200/8/2786 http://www.jimmunol.org/ References This article cites 51 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/200/8/2786.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Contribution of Adipose-Derived Factor D/Adipsin to Complement Alternative Pathway Activation: Lessons from Lipodystrophy

Xiaobo Wu,* Irina Hutson,† Antonina M. Akk,* Smita Mascharak,‡ Christine T. N. Pham,*,x Dennis E. Hourcade,* Rebecca Brown,{ John P. Atkinson,* and Charles A. Harris†,‖

Factor D (FD) is an essential component of the complement alternative pathway (AP). It is an attractive pharmaceutical target because it is an AP-speciﬁc protease circulating in blood. Most components of the complement activation pathways are produced by the liver, but FD is highly expressed by adipose tissue. Two critical questions are: 1) to what degree does adipose tissue contribute to circulating FD levels and 2) what quantity of FD is sufﬁcient to maintain a functional AP? To address these issues, we studied a novel mouse strain

with complete lipodystrophy (LD), the fld mouse with partial LD, an FD-deficient mouse, and samples from lipodystrophic patients. FD Downloaded from was undetectable in the serum of LD mice, which also showed minimal AP function. Reconstitution with purified FD, serum mixing experiments, and studies of partial LD mice all demonstrated that a low level of serum FD is sufficient for normal AP activity in the mouse system. This conclusion was further supported by experiments in which wild-type adipose precursors were transplanted into LD mice. Our results indicate that almost all FD in mouse serum is derived from adipose tissue. In contrast, FD levels were reduced ∼50% in the sera of patients with congenital generalized LD. Our studies further demonstrate that a relatively small amount of serum FD is sufficient to facilitate significant time-dependent AP activity in humans and in mice. Furthermore, this observation highlights the http://www.jimmunol.org/ potential importance of obtaining nearly complete inhibition of FD in treating alternative complement activation in various autoimmune and inflammatory human diseases. The Journal of Immunology, 2018, 200: 2786–2797.

omplement is an important arm of innate immunity binding to a microbial surface, it engages the protease precursor against bacteria and viruses and its activated products also (zymogen) factor B (FB) to form C3bB. This bimolecular complex C serve as a bridge between innate and adaptive immunity. is cleaved by factor D (FD), a serine protease unique to AP. The Three distinct but related pathways are responsible for complement cleavage results in C3bBb, known as the AP C3 convertase, which activation: the classical pathway (CP), the alternative pathway is stabilized by properdin (P), an AP-specific positive regulator. FD (AP), and the lectin pathway. All three enzymatic pathways itself is cleaved from a pro-FD zymogen to a mature FD. This by guest on September 25, 2021 converge on the cleavage of C3. This results in the generation of cleavage is mediated by members of the mannose-binding protein- proinflammatory complement fragment C3a and a major opsonin associated serine protease (MASP) family. Mice deficient in both C3b. The latter also serves as gateway to the membrane attack MASP1 and MASP3 only have pro-FD in the serum and no AP complex and a substrate to engage the AP’s amplification loop. In activity, suggesting that MASP1/3 serve as proteases to cleave pro- the CP, C1, via its C1q subcomponent, binds Ab–Ag complexes, FD into FD (1). MASP3 is thought to be the major pro-FD con- thus attacking foreign agents. In the lectin pathway, a plasma vertase whereas MASP1 is the minor convertase (2–4). lectin, such as Abs ficolins, collectins, or mannose-binding lectin, Owing to its low concentration in human serum [2–4 mg/ml (5, 6) binds to the microbial surface. In the AP, upon C3b’s covalent and our data in this study] and highly selective function, FD is a

*Division of Rheumatology, Department of Medicine, Washington University School analyzed and interpreted data; and C.T.N.P., D.E.H., R.B., and J.P.A. provided re- of Medicine, St. Louis, MO 63110; †Division of Endocrinology, Metabolism and sources, interpreted data, and revised the manuscript. Lipid Research, Department of Medicine, Washington University School of Medi- The contents do not represent the views of the U.S. Department of Veterans Affairs or cine, St. Louis, MO 63110; ‡Department of Medicine, David Geffen School of x the U.S. Government. Medicine at University of California, Los Angeles, Los Angeles, CA 90095; Section of Rheumatology, Department of Medicine, St. Louis Veterans Affairs Medical Cen- Address correspondence and reprint requests to Dr. Charles A. Harris or Dr. Xiaobo ter, St. Louis, MO 63106; {Diabetes, Endocrinology, and Obesity Branch, National Wu, Division of Endocrinology, Metabolism and Lipid Research, Department of Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Medicine, Washington University School of Medicine, 660 South Euclid Avenue, Health, Bethesda, MD 20814; and ‖Section of Endocrinology, Department of Med- Box 8127, Saint Louis, MO 63110 (C.A.H.) or Division of Rheumatology, Depart- icine, St. Louis Veterans Affairs Medical Center, St. Louis, MO 63106 ment of Medicine, Washington University School of Medicine, 660 South Euclid Avenue, Box 8045, St. Louis, MO 63110 (X.W.). E-mail addresses: harrisc@wustl. ORCIDs: 0000-0002-6703-2272 (X.W.); 0000-0003-1127-3699 (C.T.N.P.); 0000- edu (C.A.H.) or [email protected] (X.W.) 0002-2589-7382 (R.B.); 0000-0002-2514-3441 (J.P.A.); 0000-0003-1815- 5046 (C.A.H.). Abbreviations used in this article: AGL, acquired generalized LD; AMD, age-related macular degeneration; AP, alternative pathway; BAT, brown adipose tissue; BSCL, Received for publication December 1, 2017. Accepted for publication February 15, Berardinelli–Seip congenital LD; CGL, congenital generalized LD; CP, classical 2018. pathway; CVF, cobra venom factor; DIX, dexamethasone, insulin, and 3-isobutyl- This work was supported by National Institutes of Health Grants R01 DK106083 1-methylxanthine; E, embryonic day; FB, factor B; FD, factor D; ﬂd, fatty liver (to C.A.H.), 5 R01 AI041592, U54 HL112303, and 1 P30AR048335 (to J.P.A. and dystrophy; FPL, familial partial LD; hFD, human FD; LD, lipodystrophy; LMNA, X.W.), R01 AR067491 (to C.T.N.P.), and R01 AI051436 (to D.E.H. and C.T.N.P.), lamin A; MASP, mannose-binding protein-associated serine protease; MEF, mouse and by the Intramural Research Program of the National Institute of Diabetes and embryonic ﬁbroblast; P, properdin; PNGase, peptide-N-glycosidase; PPAR, peroxi- Digestive and Kidney Diseases (to R.B.). some proliferator-activated receptor; RT, room temperature; WAT, white adipose tissue; WT, wild-type. X.W. and C.A.H. performed experiments, analyzed and interpreted data, and wrote and revised the manuscript; I.H., A.M.A., and S.M. performed experiments and Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701668 The Journal of Immunology 2787

strong candidate for pharmaceutical approaches to block the pow- erful AP and its feedback loop for the treatment of numerous inflammatory diseases such as atypical hemolytic uremic syndrome (7, 8), paroxysmal nocturnal hemoglobinuria (8), and age-related macular degeneration (AMD) (9–13). Abs to FD can be used to neutralize AP activity in mice (14) and more recently have been developed for the treatment of patients with AMD (15, 16). An mAb to FD was shown to block AP in primates without affecting the CP. AP components FB and C3 in blood are largely produced by the liver but also locally by bone marrow–derived and many other cell types (17). FD is also known as adipsin because it was inde- pendently identified as an abundant transcript in 3T3-L1 adipocytes (18), but it was soon identified as identical to FD (19, 20). In addition to the high level of mRNA expression of adipsin/FD in adipose tissue, adipsin/FD expression has been described in other tissues, including muscle and lung in humans (19) and the sciatic nerve (21) as well as by the adrenal gland and ovary in mice (22). Spiegelman and colleagues (23–26) went on to demonstrate that

paradoxically adipsin mRNA is decreased in genetic and chemical Downloaded from models of obesity. More recently, they demonstrated that adipsin plays a role in pancreatic b cell function (27). We have assessed FD levels and AP activity in a variety of lipodystrophic mice. These data, along with those derived from a series of dilution and serum-mixing experiments, have enabled us

to determine that little FD is required for AP activity. In other http://www.jimmunol.org/ words, the “safety factor” for FD appears to be high. These results have clinical implications for the treatment of AP-mediated disease with FD-blocking Abs or small FD inhibitory molecules (28).

Materials and Methods Mice Lipodystrophic mice were created by crossing adiponectin-Cre mice (29) with homozygous lox-stop-lox–ROSA–diphtheria toxin A mice (30). Of note, we observed that these lipodystrophic mice die when born at room by guest on September 25, 2021 temperature (RT) and to survive must be maintained at thermoneutrality (30˚C) until weaning age. Following weaning, animals were maintained in a temperature-controlled room (22˚C) on a 12-h light/dark cycle. FD2/2, FB2/2,P2/2, and C32/2 mice have been described (31–34). Animal work was performed according to the policies of the Animal Studies Committee at Washington University School of Medicine in St. Louis. Mice were analyzed under approved protocols and were provided appropriate care while undergoing research, which complies with the standards in the Guide for the Use and Care of Laboratory Animals and the Animal Welfare Act. Mouse embryonic fibroblast differentiation Primary mouse embryonic fibroblasts (MEFs) were prepared from wild- type (WT) C57/BL6 embryonic day (E)14 embryos as described (35). Cells were allowed to become confluent and then differentiated 3 d later. White adipocytes were generated by using a mixture of 1 mM dexamethasone, 5 mg/ml insulin, and 500 mM 3-isobutyl-1-methylxanthine (DIX) mixture for 3 d followed by insulin alone for 3 d and then base media (high-glucose DMEM plus 10% FBS) for 3 d such that adipocytes are harvested 9 d after differentiation. Brown adipocytes were generated by adding the thiazolidinedione troglitazone (15 mM) to the white adipocyte mixture (DIXg mixture for 3 d and insulin-g for 3 d) (35). All adipocytes were harvested on day 9 of differentiation with radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors. We have previously demonstrated that cells differentiated with DIX express white adipose tissue (WAT) markers and not brown adipose tissue (BAT) markers, whereas cells differentiated with DIXg express BAT markers FIGURE 1. Western blot analysis of mouse FD in serum and adipose without suppressing WAT markers (S. Mascharak and C.A. Harris, un- tissue. (A) Untreated serum samples from WT mouse (0.3 ml) and also of published observations). recombinant mouse FD (rmFD; 30 ng) were treated with PNGase F. (B) Western blot of mouse FD in various adipose tissues, including epididymal WAT (EWAT), inguinal WAT (IWAT), and BAT. (C) Western blot of protocol for 9 d (DIXg9, lane 3), and 4) undifferentiated MEFs at the 1) undifferentiated MEFS prior to differentiation (UN0, lane 1), 2) conclusion of the differentiation period (UN9, lane 4). (D) Western blot of MEFs differentiated into white adipocytes with the DIX protocol for mouse FD in various fat tissues (EWAT, IWAT, and BAT) before and after 9 d (DIX9, lane 2), 3) MEFs differentiated into brown adipocytes with DIX treatment with PNGase F. These results demonstrate that mouse FD, both in plus the PPAR-g agonist troglitazone (DIXg) (Figure legend continues) serum and adipose tissues, is highly and variably N-linked glycosylated. 2788 FACTOR D/ADIPSIN AND LIPODYSTROPHY Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 2. Lipodystrophic mice. (A) WT (left) and LD (right) littermates. Note interscapular defect in LD mouse. (B) Upper, Western blot for adiponectin; lower, loading control (H chain). Pinned view of WT (C) and LD (D) mouse showing absence of inguinal fat in LD. (E and F) Skinned WT and LD P8 mice showing complete lack of adipose tissue in LD mice. H&E staining of newborn postnatal day 0 (P0) WT (G) and LD (H) mice demonstrating absence of BAT in LD. H&E staining of liver in WT (I) and LD (J) mice. Oil Red O staining of WT (K) and LD (L) mice. Note the hepatic steatosis in LD mice. White arrowheads point to adipose depots in WT mice (missing in LD mice). Black arrowheads point to brown adipose in WT mice. In LD mice, black arrowheads point to where BAT should be, but there is only a tissue plane between skeletal muscles. (G, H, K, and L) Original maginiﬁcation 310. (I and J) Original magniﬁcation 340.

Fat transplant experiments 53 buffer, and 2.5 ml of denaturant prior to being heated at 100˚C for 5 min. After cooling for 10 min and then adding 2.5 ml Triton X-100 and Primary MEFs were prepared from WT histocompatible C57/BL6 E14 2.0 ml of PNGase F to the reaction, the sample was incubated for 3 h at embryos as described (35). MEFs were injected s.c. at the sternum as 37˚C. Reaction solution (50 ml) was then mixed with 50 mlofSDS reported (36). Mice were sacriﬁced 2 mo after transplantation and sera sample buffer containing reducing reagent (Sigma-Aldrich, St. Louis, were used to measure FD levels and AP activity. MO). After heating for 60˚C for 5 min, 30 ml of solution, equivalent to Peptide-N-glycosidase assays 0.3 ml of serum, was added and analyzed by SDS-PAGE. LPS assay for AP activity in plasma (37) Recombinant mouse FD from Sino Biological (Beijing, China) and freshly isolated mouse serum were used in assays to remove N-linked glycosylated Plasma samples were harvested from mice using sterile technique. ELISA sugars by peptide-N-glycosidase (PNGase) F (QA-Bio, Palm Desert, CA). plates (catalog no. 3855; Thermo Fisher Scientiﬁc, Waltham, MA) were Following the manufacturer’s protocol, 1 ml of serum and various amounts coated with LPS (2 mg per well, catalog no. L2762; Sigma-Aldrich) at 4˚C of recombinant mouse FD were mixed with 30 mlofH2O, 10 mlof overnight. After washing three times in buffer (PBS/0.05% Tween 20) and The Journal of Immunology 2789 Downloaded from http://www.jimmunol.org/

FIGURE 4. Deﬁciency of AP activity in sera of LD mice. (A) LPS-based in vitro binding assay in sera of WT (black, n = 8), LD (gray, n = 7), and by guest on September 25, 2021 FB2/2 (white, n = 4) mice. Left, assay performed with 20% serum; right, assay performed with 10% serum. Samples normalized to WT 20% sera result equals 100% activity. (B) Rabbit RBC-based hemolysis assay on sera from WT (n = 14), LD (n = 9), FD2/2 (n = 8), or FB2/2 (n = 3) mice. Data normalized to WT equals 100% activity. These functional assays demonstrate a severe defect in AP activity of LD mice.

well). The reaction was stopped with 50 mlof1MH2SO4, and the OD of samples was measured at 450 nm. Similar experiments were performed with normal human serum and FD-depleted human serum supplemented with purified human FD (CompTech, Tyler, TX). The primary and secondary Abs used in the LPS assay with human serum are: mouse anti- human C3d (no. HYB 005-01-02; Thermo Scientific) and HRP donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories), respectively. Rabbit RBC assay for AP activity FIGURE 3. Absence of FD in LD mice. (A) Western blot analysis of complement components (FD, C3, FB, and P) in sera of WT and LD mice. A standard rabbit RBC hemolysis assay was employed to measure AP activity of m FD was undetectable whereas FB was increased ∼2-fold in the serum of LD mouse serum (34). Rabbit RBCs (1 l) (Colorado Serum Company, Denver, CO) were placed in AP buffer (GVB with 20 mM MgCl2 and8mMEGTA). mice. Serum C3 and P levels were similar between WT and LD mice. A Following centrifugation, the pellet was resuspended in the AP buffer and ali- small amount of C3 a2, a degradation fragment of complement activation quots of 20% serum from lipodystrophy (LD) and various complement-deficient produced during sample collection, was observed in some samples. NS, mice as well as human serum samples were added as described. After a 2 h nonspecific band. (B) Quantification of (A) by densitometry. *p , 0.05. incubation at 37˚C, released hemoglobin was measured at an OD of 405 nm. Lysis of rabbit RBCs in water served as the positive control whereas rabbit blocking with BSA (250 ml per well, 1% in PBS buffer), diluted mouse RBCs in AP buffer served as the negative control. Hemolysis was determined 2+ by an OD ratio, that is, (20% serum with RBC in AP buffer 2 20% serum plasma (1:5 or 1:10 in Mg -EGTA buffer) was incubated on an ELISA 2 plate at 37˚C for 1 h (50 ml per well). The plates were washed three times without RBC in AP buffer)/(RBC in water RBC in AP buffer). To account for minor differences in hemolysis between experiments, in each experiment the in washing buffer and then goat anti-mouse C3 Ab (100 ml per well, values were normalized to WT sera being 100% hemolysis. 1:4000 dilution in 1% BSA in PBS buffer; MP Cappel; MP Biomedicals, Santa Ana, CA) was added for 1 h. Wells were washed three times as Serum mixing experiments above and incubated with HRP-conjugated anti-goat IgG (100 ml per well, 1:2000 dilution in 1% BSA in PBS buffer; Jackson ImmunoResearch As sera makes up 20% of the rabbit RBC hemolysis assay (above), we kept Laboratories, West Grove, PA) for 1 h. After three washes, substrate re- the total serum concentration the same, but we varied the contribution of agent (R&D Systems, Minneapolis, MN) was added for 10 min (100 ml per WT serum and serum from knockout mice (C3, FB, FD, or P). For human 2790 FACTOR D/ADIPSIN AND LIPODYSTROPHY Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. Rescue of AP defect in LD and FD2/2 mice by puriﬁed human FD. (A) Rabbit RBC-based hemolysis assay was performed to assess AP activity in WT (white), LD (gray), and FD2/2 (black) sera (n =3 for all groups). Various quantities of FD were added to the reaction mixture. Human FD (100 ng/ml) rescued AP defect in the LD and FD2/2 mice. All values were normalized to WT equals 100%. (B) RBC hemolytic assays were with sera from WT, C3+/2,FB+/2, and FD+/2 mice (n = 3). All values were normalized to WT equals 100%. *p , 0.05. serum mixing experiments, normal human serum was mixed with serum FIGURE 6. Serum mixing experiments to assess the relative require- depleted of C3 or FD (CompTech). ments of FD (red triangle), C3 (black inverted triangle), FB (black square), Western blot or P (black circle) in AP activation. Hemolytic assays were performed at a ﬁnal concentration of 20% serum with different percentages of mixtures Fresh serum from mice was employed to measure C3, FB, P, or FD by between WT and knockout sera. For the sake of clarity, 20% WT sera in Western blotting. Goat anti-M C3 (MP Biomedicals), goat anti-Hu FB A (CompTech), rabbit anti-M P (37), sheep anti-M FD (R&D Systems), and the assay then correlates to 100% WT. ( ) High-range WT sera (WT sera B C rabbit anti-Hu FD (Abcam, Cambridge, MA) were incubated with the is 12.5–50% of total sera), ( ) mid-range (2.5–10%) WT sera, and ( )low- membranes for 2 h at RT or overnight at 4˚C. Secondary HRP-conjugated range (0.05–5%) WT sera are shown. P levels affect AP modestly in the rabbit anti-goat IgG (Sigma-Aldrich), goat anti-rabbit IgG (GE Healthcare 20% serum assay system. Whereas FD, FB, and C3 are all required for AP UK), donkey anti-sheep IgG (R&D Systems), or rabbit anti-human IgG activity, the quantity of FD required is much lower than FB and C3. Note (Jackson ImmunoResearch Laboratories) was added for 1 h at 37˚C. that 100% WT and 0% WT (100% knockout) were performed with each Membranes were developed with a SuperSignal West kit (Pierce). experiment. Data are plotted with logarithmic x-axis to show spread of Cobra venom factor treatments values with break to include 0% WT data. For in vitro experiments, 10 ml of serum, 10 mlof30mg/ml cobra venom factor (CVF; Quidel, San Diego, CA) and 10 ml of AP buffer (GVB with Human samples 20 mM MgCl2 and 8 mM EGTA) were incubated at 37˚C for 30 min. For in vivo experiments, 30 mg of CVF was injected i.p. into the mice and Patients were enrolled in a study to evaluate the effects of recombinant serum was harvested 2 h after the CVF injection. All samples were sub- human methionyl leptin (metreleptin) therapy on LD (https://clinicaltrials. jected to Western blots under reducing conditions. gov/ct2/show/NCT00025883). We only used baseline sera samples (before The Journal of Immunology 2791 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 7. FD level and AP activity in fld mice. (A) Western blot analysis for complement components in fld mice. (B) Quantification of (A). (C) Titration of FD level in fld mice. (D) Rabbit RBC-based hemolytic assay to measure AP activity in fld mice. Although there was ∼30% of FD remaining in fld mice, fld sera (white) had ∼80% of AP activity compared with WT mice (black); nonsignificant, n = 3 per group. NS, nonspecific band. *p , 0.05. leptin treatment) for this study, which was conducted at the Clinical Center full-length isoform was observed in whole-cell lysates from in- of the National Institutes of Health and was approved by the Institutional guinal and epididymal WAT as well as BAT (Fig. 1B). We also Review Board of the National Institute of Diabetes and Digestive and saw similar sized full-length isoforms in whole-cell lysates of Kidney Diseases. Written informed consent was obtained from all par- ticipants. Samples were also collected from healthy volunteers with in- MEFs that had been differentiated into white as well as brown formed consent under a protocol approved by the Washington University adipocytes, whereas no FD was observed in undifferentiated Human Studies Committee. Human FD was determined by an ELISA kit MEFs (Fig. 1C). Treatment of sera or recombinant mouse FD with from R&D Systems according to the manufacturer’s protocol as well as by PNGase F resulted in a single, 25-kDa deglycosylated isoform Western blots with anti-human FD Ab from Abcam. (Fig. 1A). This also occurred when whole-tissue lysates from Results visceral epididymal WAT, superficial inguinal WAT, and BAT (which have different physiological roles) were treated with Mouse FD is synthesized as a glycosylated protein by white PNGase F (Fig. 1D). and brown adipocytes and adipose tissue Both endogenous FD of mouse serum and recombinant mouse FD Lipodystrophic mice were a mixture of glycosylated isoforms with two prominent We set out to create lipodystrophic mice by crossing two publicly bands at an Mr between 37 and 50 kDa (Fig. 1A). A similar sized available lines (29, 30). When adiponectin-cre mice were crossed 2792 FACTOR D/ADIPSIN AND LIPODYSTROPHY

AP activity in LD mice We next asked whether FD could be detected in the plasma of LD mice. Western blotting revealed FD in the serum of WT mice, but FD was undetectable in the plasma of LD mice (Fig. 3). We also examined the relative abundance of the other AP complement proteins in serum. C3 and P levels were similar in WT and LD mice, but, interestingly, the FB concentration was approximately twice the level in LD compared with WT mice (Fig. 3). We subsequently used two functional assays to determine the activity of the AP activity in the LD mouse. There was no detectable AP in LD plasma in an LPS-coated microtiter plate assay whereas WT plasma demonstrated robust activity (Fig. 4A). In this assay, plasma is added to LPS-coated wells and then the quantity of deposited C3 fragments is determined. We performed a second AP assay testing the ability of serum to lyse rabbit RBCs. In this experiment LD sera AP activity was 15% of the WT activity (Fig. 4B). We followed these experiments by investigating whether purified human FD could rescue the AP defect in sera from LD mice and Downloaded from FD2/2 mice. FD (20 ng/ml) rescued ∼50% of the AP defect in both LD and FD-null mice and 100 ng/ml FD completely rescued the AP defect in sera from both LD and FD2/2 mice (Fig. 5A). These data further indicate that LD mice have virtually no FD and that relatively small amounts of FD are sufficient to rescue an FD deficit. We next examined sera from mice heterozygous for C3, FB, or http://www.jimmunol.org/ FD. There was a modest decrease in AP activity of C3 (63.3 6 22.1%, p , 0.05 versus WT) and FB heterozygous mice (76.7 6 18.8%, p , 0.05 versus WT), but no difference between FD heterozygous mice (109.3 6 4.3%) and WT mice (100 6 18.9%, Fig. 5B). To more directly investigate the relationship between mouse FD levels and AP activity, we conducted mouse serum mixing ex-

periments. In hemolysis assays, the total amount of serum was held by guest on September 25, 2021 FIGURE 8. FD concentrations and AP activity profile in LD mice following an adipose tissue transplant. (A) Western blot analysis of FB and constant at 20% (i.e., 20 ml of sera in a 100-ml RBC lysis reaction) FD in mice. In the titration experiments, FD levels in LD mice receiving a but the contribution of WT serum and mutant serum was altered. 2/2 fat transplant were estimated to be ∼1–5% of the WT mice; FB served as a This was accomplished by mixing WT sera with that from FD 2 2 2 2 2 2 loading control. (B) Rabbit RBC-based hemolytic assay to measure AP mice, P / ,FB / ,orC3 / mice. The absence of P only had a activity in WT, LD, and LD mice (n = 3–5) that had received preadipocyte small effect on the AP activity as measured by RBC hemolysis 2/2 2/2 transplant (LD + Fat). FD and FB served as controls. *p , 0.05. assay (Fig. 6). Reduction of FB or C3 in the reaction by mixing WT serum with either FB2/2 or C32/2 sera resulted in a linear relationship between the amount of FB or C3 and AP activity. to homozygous lox-stop-lox–diphtheria toxin A mice and pups However, FD showed a strikingly different relationship. When the were delivered at RT, adiponectin-cre+ mice were not found at FD concentration was lowered to 50, 25, or 12.5% of the WT 2/2 Mendelian frequency (3 of 46 mice were Cre+ and lipodystrophic, levels (by mixing with FD sera), the AP activity remained p , 0.05 by x2 test). To assess whether this was due to nonspecific normal (Fig. 6A). Experiments in which mixing serum at a lower leakage of adiponectin-cre in another tissue or whether it was a range of WT sera showed that 2.5% of normal FD levels main- specific functional consequence due to the loss of adipose tissue, tained full AP activity (Fig. 6B). A further mixing experiment we postulated that the latter might be rescued by allowing mice to showed that a FD level of 0.5% (10 ng/ml using our measured be born in a thermoneutral setting; that is, the reduced viability value of FD of 10 mg/ml in mouse serum) supported 43% of AP was due to the loss of BAT-mediated thermoregulation and re- activity (Fig. 6C). sultant hypothermia. The cross was repeated with the dam moved To gain additional insight into the relationship among adipose from RT to thermoneutrality once they appeared pregnant (∼E14). mass, FD levels, and AP activity, we examined an additional mouse Mutant double-transgenic mice were now born at the expected strain with partial LD, fatty liver dystrophy (fld). Fld mice have Mendelian frequency. Subsequently, we found that pregnant mice ∼80% loss of adipose tissue (38). FD levels were reduced by carrying mutant progeny could be moved from RT to thermo- ∼70% in sera of fld mice (compared with WT mice, Fig. 7A, 7B), neutrality up until the night before parturition without loss of whereas levels of other complement components, C3, FB, and P, viability in lipodystrophic pups (27 of 62 progeny Cre+, not sta- were similar to WT mice (Fig. 7A, 7B). Titration of plasma from tistically different from the Mendelian expected 50%). Double- fld mice also showed that FD levels were ∼30% of WT (Fig. 7C). transgenic lipodystrophic mice can be recognized soon after Despite only having ∼30% of normal FD levels, serum from fld birth due to an interscapular defect normally containing BAT mice reconstituted 77.5 6 8.1% of WT AP activity (nonsignificant (Fig. 2A). LD mice had undetectable adiponectin (Fig. 2B), compared with WT) in the rabbit RBC hemolysis assay (Fig. 7D). grossly absent WAT (Fig. 2C–F) and BAT (Fig. 2G, 2H), and The other approach we used to probe the relationship among massive hepatic steatosis (Fig. 2I–L) adipose mass, FD levels, and AP activity was to perform rescue The Journal of Immunology 2793 Downloaded from http://www.jimmunol.org/

FIGURE 9. CVF leads to C3 cleavage in sera of LD mice. (A) CVF induces cleavage in vitro of C3 in sera of WT and LD mice, but not FD2/2 or FB2/2 mice. (B) CVF injection results in C3 cleavage in WT, LD, and FD2/2 mice, but not in FB2/2 mice. All gels were run under reducing conditions. All experiments were performed at least twice with representative results shown. NS, nonspeciﬁc band.

experiments with LD mice. We harvested MEFs from histo- demonstrated an absence of FD in the sera of FD2/2 and LD compatible C57/BL6 E14 embryos and injected the cells s.c. mice and increased FB in the sera of LD mice. under the sternum of LD mice (36). A large fat pad developed by guest on September 25, 2021 FD/adipsin in human LD at the injection site within 4 wk of injection. Mice were sac- rificed 8 wk after injection. The fat pads weighed 500–1000 We next pursued whether the absolute requirement of adipose mg, and therefore constituted 10–20% of the normal fat mass tissue for circulating FD levels observed in the mouse system of age-matched WT mice. Remarkably, many of the metabolic applied to humans. We tested circulating FD levels in 1) normal derangements (e.g., fatty liver) were rescued by the formation subjects, 2) patients with congenital generalized LD (CGL) due to of the ectopic fat pad. We were now also able to detect cir- mutations in acyl-glycerol acyltransferase 2 (n = 3) or Berardi- culating FD in LD mice that had been rescued by MEF in- nelli–Seip congenital LD (BSCL)2 (n = 1), 3) patients with familial jection. However, the FD level in transplanted mice only partial LD (FPL) due to mutations in lamin A (LMNA, n =7), represented ∼1–5% of WT circulating FD levels (Fig. 8A). and 4) patients with acquired generalized LD (AGL) (n =5)using Despite these reduced FD levels, sera from the rescued LD an ELISA. We found that FD was detectable in patients with these mice were able to support AP activity at a level of ∼30% of forms of LD, but that FD levels were significantly lower in CGL WT levels (Fig. 8B). than in controls. Specifically, CGL patients had circulating FD CVF is similar to host-derived C3b in that it can work together levels ∼50% of that in controls (Fig. 10A). Patients with FPL or with FB to trigger the AP. Consequently, we examined the ability of AGL had levels of FD between those of controls and CGL. We CVF to induce C3 cleavage (to C3b) in sera of WT, LD, FD2/2,or performed a Western blot on the sera from patients with CGL and FB2/2 mice in vitro. CVF robustly induced cleavage of C3 in WT controls and observed that FD levels were reduced in CGL patients and LD sera, whereas very little cleavage occurred in sera from compared with controls (see Fig. 10B). Our data on human FD with either FD2/2 or FB2/2 mice (Fig. 9A). The FB and FD blots Western blots showed that there was a single unglycosylated band confirmed the absence of FD in LD and FD2/2 sera and the in- with a molecular mass of ∼25 kDa. Because of the variability in fat creased FB in LD sera. mass in these patients, we created a plot of adipose mass versus FD We used the ability of CVF to induce C3 cleavage in vivo when levels and saw a linear relationship between adipose mass and FD injected into WT, LD, FD2/2,orFB2/2 mice. CVF injection into levels (Fig. 10C, R2 = 0.388). We performed the RBC hemolysis WT mice resulted in C3 cleavage. CVF injection into FB2/2 assay for AP on patient sera and observed equivalent levels of RBC mice resulted in no cleavage of C3, indicating that FB is abso- hemolysis with CGL and control sera. lutely required for C3 cleavage. Surprisingly, injection of CVF To determine whether the low concentrations of FD were also into LD or FD2/2 mice resulted in cleavage of C3, suggesting sufficient for activation of AP activity in the human system, we that FD may be dispensable for CVF-induced cleavage of performed mixing experiments between normal human sera and C3 in vivo (Fig. 9B). This observation is similar to that in ex- human sera that had been immunodepleted of either C3 or FD. periments performed when FD2/2 mice were initially de- These experiments mirrored what was seen in mice; namely, a low scribed (31). For these experiments, Western blotting again concentration of human FD was needed to activate AP (∼1% for 2794 FACTOR D/ADIPSIN AND LIPODYSTROPHY

FIGURE 10. Reduction in circulating FD levels in patients with CGL. (A) FD levels were measured by ELISA for the following groups: 1) control (black, n = 4), 2) CGL due to acyl- glycerol acyltransferase 2 or BSCL2 mutations (light gray, n = 5), 3) AGL (dark gray, n = 5), Downloaded from and 4) FPL due to mutations in LMNA or PPARG (white, n = 8). *p , 0.05. (B) Reduced FD in CGL patients observed in Western blot. p , 0.05 CGL versus controls. FD lane is 10 ng of puriﬁed human FD. Gel was run under reducing condition. HC, H chain of IgG. (C)

Scatter plot of adipose mass (kilograms) versus http://www.jimmunol.org/ FD levels (nanograms per milliliter). R2 = 0.388. d, AGL; n, FPL, unknown type; ▴, CGL, atypical Dunnigan’s; ;, CGL-BSCL1; ♦, CGL-BSCL2; s, FPL-LMNA; N, FPL, PPAR-g.(D) Mixing of normal human sera control with either FD or C3-depleted sera. Experiment was repeated three times. by guest on September 25, 2021

half-maximal activity) whereas there was more of a linear rela- min there was .90% lysis when using 400 ng/ml human FD tionship between human C3 for AP activation (Fig. 10D). (hFD; Fig. 11B). Moreover, substantial lysis was obtained at 25 ng/ml FD in the standard 30-min incubation commonly employed Requirement for FD in two quantitative assays in this assay system (Fig. 11C). We also performed a dose re- The literature from the 1970s suggests that FD is the limiting factor sponse curve for FD in the LPS microplate assay using different in AP activation (39). That view was based on rabbit hemoly- incubation times both in human (adding hFD to FD-depleted se- sis assays performed using the addition of purified human FD rum) and mouse (mixing WT and FD-null sera) systems (Fig. 11D, or human serum to substitute for diisopropylfluorophosphate- 11E). When we added various amounts of hFD back to FD- inactivated FD in human plasma (39). Those experiments depleted human serum we observed an EC50 of 76 ng/ml for the showed that adding more than physiological amounts of FD (up to activation of AP with a 30-min incubation and an EC50 of 5 ng/ml 9-fold) resulted in increased AP activity. Those experiments, with a 2-h incubation (Fig. 11D). Addition of hFD to WT serum however, were carried out with incubation times of only 5 min. We did not increase AP activity (data not shown). We also conducted have performed equivalent assays using purified human FD and similar experiments by mixing WT mouse sera with FD-null FD-depleted human serum with a similar result at 5 min incuba- mouse sera. For this experiment the total percentage of serum in tion (Fig. 11A, 11B). In contrast, at longer incubation times much the assay was held constant (20%) but the proportion of WT and less added FD was required to attain WT-level AP activity FD-null serum varied. Using our measured values of 10 mg/ml for (Fig. 11C). Note that in this lytic system employing RBCs, by 10 WT serum, the amount of FD required for 50% activation in this The Journal of Immunology 2795 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 11. Kinetics and dose response curve for human FD in the rabbit RBC hemolysis and LPS microplate assays for AP. (A–D) Performed in FD- depleted human serum reconstituted with hFD. (A) Dose response curve for hFD with a 5-min incubation. (B) Kinetics of hemolysis when using 400 ng/ml hFD. (C) Dose response curve of hFD when used with a 30-min incubation. RBC hemolysis data are expressed as percentage hemolysis. (D) Dose response curve for hFD added to FD-depleted human serum in the LPS microplate assay. (E) Dose response curve for FD mixing experiment using WT and FD2/2 mouse serum in the LPS microplate assay. assay was 1.8% (36 ng/ml) and 0.3% (6 ng/ml) for the 30-min and tissues might have the capacity to produce FD, it is the adipose 2-h incubation times, respectively (Fig. 11E). These numbers are tissue that accounts for almost all of the circulating FD in mice. quite similar to what we saw with the RBC hemolysis assay. Note that when comparing the mouse and human systems, circulating mouse FD is found at higher concentrations [10–30 mg/ Discussion ml; our data and Ref. (1)] than human FD (2–4 mg/ml). Our studies using lipodystrophic and FD2/2 mice establish that In contrast to the mouse, there were reduced (∼50%, p , 0.05) FD/adipsin is almost entirely produced from adipose tissue. FD FD levels in the sera of patients with CGL. This is most likely was not detected in LD mice. Although FD was previously de- because the patients with LD still have a small amount of adipose scribed to be highly expressed in adipose tissues, others had tissue as compared with the lipodystrophic mice that were ge- reported significant expression in nonadipose tissues such as by netically engineered to be completely devoid of adipose tissue. the sciatic nerve in mice (21) as well as by muscle and lung in Alternatively, either FD is normally secreted from nonadipose humans (19). Our experiments indicate though that although other tissues in humans, or nonadipose tissues [e.g., the liver, which 2796 FACTOR D/ADIPSIN AND LIPODYSTROPHY highly expresses peroxisome proliferator-activated receptor approach was prompted by the observation of complement system (PPAR)-g and some adipocyte markers in the lipodystrophic state] proteins being deposited in the retina of patients with AMD (48) (40) compensate to secrete FD in the lipodystrophic state. FD and then followed by the discovery that common (9–12) and rare levels were reduced to a lesser extent in the sera of patients with variants (49, 50) in complement factor H are associated with risk AGL or FPL, a state of partial loss of adipose mass. It is unclear for AMD. Future studies will determine whether FD inhibition why patients with AGL did not have as severe a reduction in FD will result in immunosuppression and opportunistic infections levels compared with CGL patients. One potential explanation is (which could be addressed prophylactically with meningococcal the fact that AGL patients retain variable amounts of visceral vaccination) (51, 52) and whether low levels of functional FD that adipose tissue whereas it is completely absent in CGL patients may survive FD inhibition are sufficient to sustain pathogenesis in (41–43). Additionally, marrow adipose tissue is retained in AGL AP-mediated disease models, especially in chronic inflammatory and absent in both CGL1 and CGL2 (41–43), the patients included conditions such as AMD. in the present study. Also, it is perhaps not too surprising that We further speculate that these data are likely to affect how we patients with CGL have detectable FD and AP activity, as an in- envision complement activation in extravascular tissue sites. Rather creased incidence of Neisseria infections is not observed in these amazingly, the results indicate that low levels of FD are sufficient to patients as has been described in patients with loss-of-function FD mediate AP activation. We propose that this process is a funda- mutations (44–46). mental mechanism by which the host responds to altered self, that Another interesting observation was that lipodystrophic mice is, cellular and tissue debris that is continuously generated (both for had increased levels of circulating FB. This is unlikely to be due intracellular and extracellular disposal). solely to a compensatory effect in the absence of FD, as it was not Following this line of reasoning, we hypothesize that this dis- Downloaded from seen in FD2/2 mice (Fig. 8). FB is an acute phase response protein posal activity was part of the original complement system. It was (17). We favor a hypothesis that the hepatic steatosis in LD mice then adapted by the host to facilitate recognition of a foreign agent. induces hepatic inflammation and this in turn stimulates hepatic With the subsequent evolutionary development of circulatory FB expression. systems, reaction speed becomes more critical. However, the AP, in A finding of note is that CVF was able to facilitate in vitro C3 particular, has maintained its ability to recognize and opsonize 2/2 cleavage to a greater extent in LD than FD sera in vitro debris. http://www.jimmunol.org/ (Fig. 9A). This may be due to a small amount of FD in sera of LD mice that is not detected by our assay systems (LPS micro- Acknowledgments plate assay, RBC hemolysis, or Western blotting). Another pos- We thank Abhimanyu Garg for assistance in genotyping patients with LD, sibility is that the increased FB in sera of LD mice accounts for Brian Finck for providing the fld mice, and Madonna Bogacki and Lorraine augmented CVF-mediated C3 cleavage. However, when CVF was Schwartz for manuscript editing. injected into WT, FD2/2, LD, or FB2/2 mice, C3 was cleaved in 2 2 2 2 WT, FD / , and LD mice, but not in FB / mice (Fig. 9B). These Disclosures data point to an FD-independent mechanism of CVF-induced C3 The authors have no financial conflicts of interest. cleavage. One possibility is that a second “compensatory” prote- by guest on September 25, 2021 ase is responsible (31). Kallikrein is a possible candidate given its similar protease activity to FD (47). References Using both mixing experiments with sera from FD2/2 mice and 1. Takahashi, M., Y. Ishida, D. Iwaki, K. Kanno, T. Suzuki, Y. Endo, Y. Homma, and T. Fujita. 2010. 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