CTRP6 promotes the macrophage inflammatory response, and its deficiency attenuates LPS-induced inflammation

Macrophages play critical roles in inflammation and tissue homeostasis, and their functions are regulated by various autocrine, paracrine, and endocrine factors. We have previously shown that CTRP6, a secreted protein of the C1q family, targets both adipocytes and macrophages to promote obesity-linked inflammation. However, the gene programs and signaling pathways directly regulated by CTRP6 in macrophages remain unknown. Here, we combine transcriptomic and phosphoproteomic analyses to show that CTRP6 activates inflammatory gene programs and signaling pathways in mouse bone marrow-derived macrophages (BMDMs). Treatment of BMDMs with CTRP6 upregulated proinflammatory, and suppressed the antiinflammatory, gene expression. We also showed that CTRP6 activates p44/42-MAPK, p38-MAPK, and NF-κB signaling pathways to promote inflammatory cytokine secretion from BMDMs, and that pharmacologic inhibition of these signaling pathways markedly attenuated the effects of CTRP6. Pretreatment of BMDMs with CTRP6 also sensitized and potentiated the BMDMs response to lipopolysaccharide (LPS)-induced inflammatory signaling and cytokine secretion. Consistent with the metabolic phenotype of proinflammatory macrophages, CTRP6 treatment induced a shift toward aerobic glycolysis and lactate production, reduced oxidative metabolism, and elevated mitochondrial reactive oxygen species production in BMDMs. Importantly, in accordance with our in vitro findings, BMDMs from CTRP6-deficient mice were less inflammatory at baseline and showed a marked suppression of LPS-induced inflammatory gene expression and cytokine secretion. Finally, loss of CTRP6 in mice also dampened LPS-induced inflammation and hypothermia. Collectively, our findings suggest that CTRP6 regulates and primes the macrophage response to inflammatory stimuli and thus may have a role in modulating tissue inflammatory tone in different physiological and disease contexts.

Macrophages play critical roles in inflammation and tissue homeostasis, and their functions are regulated by various autocrine, paracrine, and endocrine factors.We have previously shown that CTRP6, a secreted protein of the C1q family, targets both adipocytes and macrophages to promote obesitylinked inflammation.However, the gene programs and signaling pathways directly regulated by CTRP6 in macrophages remain unknown.Here, we combine transcriptomic and phosphoproteomic analyses to show that CTRP6 activates inflammatory gene programs and signaling pathways in mouse bone marrow-derived macrophages (BMDMs).Treatment of BMDMs with CTRP6 upregulated proinflammatory, and suppressed the antiinflammatory, gene expression.We also showed that CTRP6 activates p44/42-MAPK, p38-MAPK, and NF-κB signaling pathways to promote inflammatory cytokine secretion from BMDMs, and that pharmacologic inhibition of these signaling pathways markedly attenuated the effects of CTRP6.Pretreatment of BMDMs with CTRP6 also sensitized and potentiated the BMDMs response to lipopolysaccharide (LPS)-induced inflammatory signaling and cytokine secretion.Consistent with the metabolic phenotype of proinflammatory macrophages, CTRP6 treatment induced a shift toward aerobic glycolysis and lactate production, reduced oxidative metabolism, and elevated mitochondrial reactive oxygen species production in BMDMs.Importantly, in accordance with our in vitro findings, BMDMs from CTRP6-deficient mice were less inflammatory at baseline and showed a marked suppression of LPS-induced inflammatory gene expression and cytokine secretion.Finally, loss of CTRP6 in mice also dampened LPSinduced inflammation and hypothermia.Collectively, our findings suggest that CTRP6 regulates and primes the macrophage response to inflammatory stimuli and thus may have a role in modulating tissue inflammatory tone in different physiological and disease contexts.
Macrophages play important and critical roles in both the innate and adaptive immune response, as well as in tissue repair and homeostasis (1-4).They express a wide variety of cell surface receptors that allow them to sense and respond to environmental cues derived from the host or pathogens (5,6).Given their pleiotropic roles, macrophages can be functionally grouped into different subsets based on specific cell surface markers and the types of mediators they secrete (7,8).
Among the different subsets based on initial studies, the classically activated M1 macrophages are generally thought to promote inflammation by secreting proinflammatory cytokines, whereas the alternatively activated M2 macrophages dampen inflammation by producing antiinflammatory cytokines (2,7).While not mutually exclusive, M1 macrophages rely heavily on aerobic glycolysis whereas M2 macrophages primarily utilize mitochondrial oxidative metabolism to fuel their energetic needs (9)(10)(11).However, with the increasing recognition that there is a wide spectrum of macrophage functional phenotypes distinct from the classical M1 or M2, or along the continuum of M1 to M2, has led to a more nuanced appreciation of macrophage functional heterogeneity and plasticity (12)(13)(14).
In the contexts of murine models of obesity, 40 to 50% of cells within the adipose compartment are macrophages derived from both circulating monocytes and the locally proliferating resident macrophages (15)(16)(17).Although less dramatic, macrophage infiltration into different fat depots has also been noted in human obesity (18).These infiltrating and locally proliferating macrophages often promote chronic low-grade inflammation within the adipose compartment (15)(16)(17)19).This could disrupt normal adipocyte function and dysregulate the adipokine secretory profile, contributing to impaired local and systemic metabolism (20,21).However, a homeostatic role of the infiltrating macrophages has also been noted in the context of weight loss, where elevated lipid flux due to enhanced lipolysis in adipose tissue promotes the recruitment of macrophages, which then turn into lipid-laden macrophages to help buffer the rise in local lipid levels (22).
We have previously shown that C1q/tumor necrosis factor (TNF)-related protein 6 (CTRP6; also known as C1qtnf6)-a secreted protein of the C1q family (23,24)-is one of the autocrine and paracrine factors that regulates macrophage function in obesity (25).CTRP6 expression is upregulated in obesity and acts on adipocytes and macrophages to promote inflammation within fat depots (25).While both adipocytes and macrophages produce CTRP6 in the fat pads of dietinduced obese mice, a proportionally higher transcript expression is found in the nonadipocyte fraction that comprises the infiltrated macrophages (25).Mice lacking CTRP6 have reduced adipose tissue inflammation and an improved systemic metabolic profile; conversely, overexpression of CTRP6 in mice impairs insulin sensitivity (25).In chronic obesity, CTRP6-mediated inflammation has a detrimental role; however, in the context of an acute short-term (3-days) caloric overload, upregulated CTRP6 expression promotes a homeostatic inflammatory response in the fat compartment to limit excess fat storage (26).Thus, the physiological outcomes of CTRP6-mediated inflammatory responses are contextdependent.
While we know macrophages are one of the cell targets of CTRP6, the gene programs and signaling pathways directly regulated by CTRP6 in macrophages to promote a proinflammatory response has not been determined and is the focus of the present study.Combining transcriptomic and phosphoproteomic analyses with cell signaling and respirometry studies, we directly demonstrated that CTRP6 promotes a proinflammatory phenotype in bone marrow-derived macrophages (BMDMs).Consistent with our in vitro findings, BMDMs derived from Ctrp6 KO mice had lower basal and lipopolysaccharide (LPS)-stimulated inflammatory cytokine gene expression and secretion.Further, mice deficient in CTRP6 had lower circulating TNF-α levels and an attenuated hypothermic response induced by an acute LPS challenge.Collectively, these results highlight the role of CTRP6 as a physiologic modulator of tissue inflammatory tone by sensitizing and potentiating macrophage's response to inflammatory stimuli.

CTRP6 promotes a macrophage proinflammatory phenotype
To better understand how CTRP6 regulates macrophage function, we performed an unbiased transcriptomics analysis of genes that are regulated by this secreted protein in BMDMs.Bulk RNA sequencing was carried out on BMDMs stimulated with vehicle control or recombinant CTRP6 for 6 h.The short time frame allowed us to focus on differentially expressed genes (DEGs) likely to be directly regulated by CTRP6.Of the DEGs (p-value < 0.05 and log2FoldChange >/<0), there were a total of 322 genes upregulated and 271 genes downregulated in BMDMs upon CTRP6 treatment (Fig. 1A and Table S1 and  S2).Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that those DEGs were enriched for signaling pathways known to promote a proinflammatory macrophage phenotype, such as the Notch (27), FoxO (28), and mitogen-activated protein kinase (MAPK) (29,30) signaling pathways (Fig. 1B).
In addition to the classic inflammatory genes, transcripts that encode proteins involved in inflammatory MAPK signaling (e.g., Rasgrp1, Irak4, Mapk14, and Map3k11) and NF-κB signaling (e.g., Mavs, Irak4, Plpp3, and Traf3) were also upregulated in BMDMs by CTRP6 treatment (Fig. 1E).Together, these results indicate that CTRP6 treatment alters the expression of many immune-related genes, changes consistent with promoting a more proinflammatory profile in BMDMs.

CTRP6 activates the MAPK and NF-κB signaling pathways to promote macrophage inflammatory response
In parallel to our transcriptomic analysis, we also performed unbiased quantitative proteomic and phosphoproteomic analyses to determine what proximal signaling events are directly regulated by CTRP6 in BMDMs (Fig. 2A).As expected, we did not observe a significant change in BMDMs at the proteomic level after CTRP6 treatment for 30 min.Of the 6993 quantitated proteins, 6989 showed <1.2 fold change in CTRP6-treated BMDMs compared with vehicle-treated ones (Table S3) while only four proteins showed >1.2 fold change with significance of p <0.05.Thus, we chose 1.2 fold change as a cutoff for CTRP6regulated phosphorylation.Out of 11,588 quantitated phosphopeptides, the phosphorylation levels on 314 peptides were significantly changed (p < 0.05) after CTRP6 treatment (Fig. 2B), among which 262 were upregulated and 52 were downregulated (Table S4).A partial list of the upregulated and downregulated phosphoproteins is shown in Tables 1 and 2. Mapk1-, MapK3-, MAPK-activated protein kinase 2 (Mapkapk2), and TNF receptor (TNFR) associated factor family member-associated NF-κB activator (Tank) were among the phosphoproteins upregulated by CTRP6 treatment in BMDMs (Table 1).Of the phosphoproteins that were significantly changed, KEGG pathway analysis revealed an enrichment for the MAPK signaling pathway (Fig. 2C).Gene Ontology analysis also confirmed that MAPK, extracellular regulated protein kinase (ERK1 and ERK2), and p38-MAPK pathways were among the top enriched biological processes (Fig. 2C).
MAPK and NF-κB are the two major signaling pathways involved in macrophage proinflammatory activation (38)(39)(40)(41).We therefore performed immunoblot analyses to further determine whether and to what extent these signaling pathways contribute to the CTRP6-induced inflammatory response in macrophages.We observed that CTRP6 induced a rapid and robust phosphorylation and activation of ERK1/2, p38-MAPK, and c-Jun N-terminal kinase signalings in BMDMs (Fig. 3A).We also noted a rapid activation of the NF-κB signaling pathway, as shown by the time-dependent degradation and reduction of inhibitor of κB (IκBα) and the corresponding increase in p65 (RelA, a subunit of NF-κB) phosphorylation (Fig. 3A).

CTRP6 augments LPS-stimulated inflammatory response in macrophages
Macrophages express toll-like receptor 4 and respond to bacterial LPS stimulation.We first assessed whether Ctrp6 gene and protein expression are positively regulated by toll-like receptor 4 signaling, and thus may constitute a feedforward loop; alternatively, LPS may reduce the expression of Ctrp6 in a temporal manner to limit excessive inflammatory response in macrophages.We observed that LPS stimulation caused a time-dependent decline in Ctrp6 transcript and protein expression, followed by a gradual rebound, in BMDMs (Fig. 4, A and B).Specifically, the expression of Ctrp6 transcript and protein began to drop at 1 h post LPS stimulation, reached the lowest level by 6 h, followed by a gradual rebound over the next 18 h.
Next, we asked whether CTRP6 can further augment inflammatory cytokine secretion, gene expression, and signaling in macrophages stimulated with LPS.Pretreating BMDMs

Proinflammatory role of CTRP6
with CTRP6 for 1 h before stimulation with LPS significantly augmented IL-6 secretion, whereas the effects on TNF-α and monocyte chemoattractant protein-1 (MCP-1) were marginally and not biologically significant (Fig. 4, C-E).At the level of gene expression, CTRP6 pretreatment further enhanced the expression of inflammatory genes (e.g., Adgre1, Tnf, Il1b, Itgax, and Nos2) induced by LPS (Fig. 4F).At the level of signaling, we observed a significantly greater LPS-induced reduction in IκB and phosphorylation of ERK1/2, p38-MAPK, and p65 (RelA) in CTRP6-pretreated BMDMs than the LPS stimulation alone (Fig. 4, G and H).These results suggest that CTRP6 can potentiate LPS-induced activation of MAPK and NF-κB signaling pathways in macrophages.Therefore, the observed time-dependent reduction in CTRP6 expression induced by LPS (Fig. 4, A and B) suggests a potential negative feedback mechanism to prevent prolonged hyperactivation of macrophages during the course of inflammation.

CTRP6 promotes aerobic glycolysis and ROS production in macrophages
A now well-established immunometabolism paradigm is that proinflammatory macrophage activation shifts the cell metabolism toward aerobic glycolysis (10,42,43).Because  CTRP6 promotes a proinflammatory macrophage phenotype, we therefore assessed whether CTRP6 also induces a shift in energy metabolism toward aerobic glycolysis in BMDMs.To help regenerate NAD + needed for ongoing aerobic glycolysis, pyruvate (the end product of glycolysis) gets reduced to lactate and excreted from cells (44).Treatment of resting BMDMs with CTRP6 resulted in greater lactate secretion (Fig. 5A).LPS stimulation appeared to maximally increase lactate secretion from BMDMs, and the cotreatment with CTRP6 did not further elevate lactate secretion (Fig. 5A).
The rate of glycolysis can be inferred from the measurement of extracellular acidification rate (ECAR) (45).Consistent with greater lactate secretion, CTRP6 treatment significantly increased ECAR in resting BMDMs, and was not further elevated by costimulation with LPS (Fig. 5B).In contrast to increased rate of glycolysis, mitochondrial oxidative metabolism was reduced in CTRP6-treated BMDMs (Fig. 5, C and  D).LPS stimulation maximally reduced mitochondrial oxidative metabolism in BMDMs, and cotreatment with CTRP6 did not further suppress oxidative metabolism (Fig. 5, C and D).Consistent with the functional data, genes involved in glycolytic flux were upregulated whereas genes involved in mitochondrial oxidative metabolism downregulated in BMDMs treated with CTRP6 (Fig. 5E).
In addition to secreting inflammatory cytokines, proinflammatory macrophages also generate reactive oxygen species (ROS) which serves as microbicidal agents in host defense.In addition to the NADPH oxidase system (46), it is known that proinflammatory macrophages also have elevated mitochondrial ROS production (47,48).We observed that CTRP6 treatment significantly increased mitochondrial ROS production in BMDMs as measured by MitoSOX staining (Fig. 5F).Since LPS maximally suppressed mitochondrial oxidative metabolism (Fig. 5D), the ability of CTRP6 to promote mitochondrial ROS production was lost in the presence of LPS (Fig. 5F).Collectively, these results indicate that CTRP6 induces a metabolic shift from oxidative metabolism toward aerobic glycolysis and increases mitochondrial ROS production in macrophages, both of which are consistent with CTRP6 promoting a proinflammatory macrophage phenotype.

CTRP6 deficiency attenuates LPS-induced inflammation and hypothermia
Next, we used BMDMs and mice deficient in CTRP6 to further confirm and extend our findings.At resting state, BMDMs derived from Ctrp6 KO mice had lower expression of proinflammatory genes, Il6 and Il1b, and higher expression of the antiinflammatory gene Il10 (Fig. 6A).In response to LPS stimulation, CTRP6-deficient BMDMs had markedly reduced expression of proinflammatory genes (e.g., Tnf, Ccl2, Il6, and Il1b) and increased expression of the antiinflammatory gene Arg1 relative to WT BMDMs (Fig. 6B).Similar to CTRP6deficient BMDMs, we also observed an attenuated proinflammatory gene expression at baseline and in response to LPS stimulation in alveolar macrophages (AMs) isolated from the lung and Kupffer cells isolated from the liver of Ctrp6-KO mice (Fig. S1).In accordance, CTRP6-deficient BMDMs also showed an attenuated LPS-induced phosphorylation of Erk1/2 and p38-MAPK, and to a much lesser extent of p65 (Fig. 6, C and D).The protein levels of IκB were higher before LPS stimulation, and remained higher for the first 60 min after LPS stimulation in CTRP6-deficient BMDMs.
Secretion of inflammatory cytokines (TNF-α, IL-6, and MCP-1) was also significantly reduced in CTRP6-deficient BMDMs compared to WT BMDMs upon LPS stimulation (Fig. 7A).Thus, deletion of Ctrp6 gene reduces the macrophage inflammatory response whereas recombinant CTRP6 treatment promotes the opposite phenotype.Lastly, we sought to determine whether CTRP6 deficiency protects mice from LPS-induced inflammation and hypothermia.Mice lacking CTRP6 had significantly lower serum TNF-α levels relative to WT controls after an LPS challenge (Fig. 7B).Serum IL-1β, IL-6, and MCP-1 levels, however, were not significantly different between genotypes (data not shown).In addition, loss of CTRP6 also significantly attenuated hypothermia induced by LPS challenge (Fig. 7C).Consistent with the physiological data, transcript expression of Tnf and Il1b was also significantly lower in the liver of Ctrp6 KO mice relative to WT controls after an LPS challenge (Fig. 7D).Together, these results indicate that CTRP6 deficiency dampens LPS-induced systemic inflammation in mice.

Discussion
Using a combination of unbiased and targeted approaches, we provided new insights concerning the direct action of CTRP6 on macrophages.We showed that CTRP6 engages the MAPK and NF-κB signaling pathways in macrophages to drive the expression of proinflammatory gene expression and cytokine production and secretion.In resting BMDMs, CTRP6 treatment alone was sufficient to promote the expression of proinflammatory genes, and induce a metabolic shift from oxidative metabolism toward aerobic glycolysis, and enhanced mitochondrial ROS production.Pretreatment with CTRP6 also sensitized and potentiated BMDMs response to LPS.In contrast, BMDMs lacking CTRP6 had significantly reduced LPS-induced proinflammatory gene expression and cytokine secretion.Crucially, mice lacking CTRP6 had attenuated systemic inflammation in response to endotoxemia, as indicated by reduced serum TNF-α and attenuated LPS-induced hypothermia.Collectively, these results along with our previous studies (25,26) extend and affirm the autocrine and paracrine role of CTRP6 in regulating macrophage function.Our findings also suggest a potential role for CTRP6 in modulating the inflammatory tone in local tissue environment under various physiological and pathophysiological states (e.g., short-term caloric surplus, infection, and chronic obesity).
In a short-term caloric overload model (3-days high-fat feeding), the Ctrp6 transcript is rapidly upregulated in the adipose tissue (26).This occurred prior to any significant infiltration of macrophages into the fat compartment.In this physiological context, the inflammatory response mediated by the locally produced CTRP6 presumably acts on adipocytes Proinflammatory role of CTRP6 and stromal vascular cells (including resident adipose tissue macrophages) to limit excess fat accumulation, thus serving a homeostatic role (26).Such homeostatic, rather than pathological, inflammatory response in the early phase of caloric surplus is well-documented (49,50).However, in the setting of obesity induced by chronic high-fat feeding, close to half of the cells in the adipose compartment in mice are comprised of macrophages (7,15,17).In this context, the direct action of CTRP6 on adipose tissue macrophages represents an important axis contributing to low-grade chronic inflammation in the fat pads.In addition, obesity is known to induce gut dysbiosis and leakiness, leading to increased circulating LPS (51,52).Based on the data presented in this study, it is likely that in obesity CTRP6 acts in concert with LPS derived from the gut bacteria to further exacerbate adipose tissue dysfunction, underscoring its potential modulatory role in priming adipose tissue macrophages to inflammatory stimuli.Consistent with this, mice lacking CTRP6 have reduced adipose tissue inflammation and improved systemic insulin sensitivity (25).
We showed that pretreatment of BMDMs with CTRP6 enhances LPS-stimulated proinflammatory signaling and cytokine secretion.Conversely, CTRP6-deficient BMDMs had a significantly dampened LPS-induced proinflammatory response.Interestingly, we also noted that acute LPS treatment caused a time-dependent suppression of CTRP6 expression in BMDMs, followed by a gradual rebound at 24 h.Since CTRP6 alone or in combination with LPS promotes the macrophage proinflammatory response, the temporal suppression of CTRP6 expression by LPS suggests a potential negative feedback mechanism to prevent prolonged macrophage hyperactivation, as an uncontrolled inflammatory response is detrimental.In contrast to this potential negative feedback mechanism by LPS, we previously showed that CTRP6 treatment of macrophages can upregulate Ctrp6 expression, resulting in positive reinforcement (25).However, what signal(s) terminates this positive feedback once CTRP6 action is initiated remains to be identified.This signal(s)-in the form of a secreted factor or metabolite-likely exists, as we have previously noted that Ctrp6 expression in obesity can be downregulated back to normal levels if mice are subjected to a diet reversal (i.e., switching from a high-fat to a low-fat diet after obesity induction) (26).Collectively, these observations indicate that Ctrp6 expression in macrophages is subjected to both positive and negative regulations, and dysregulated CTRP6 expression may contribute to pathology in diseased settings.
The CTRP6-induced metabolic shift from oxidative metabolism toward aerobic glycolysis in BMDMs is consistent with the metabolic phenotype of proinflammatory macrophages reported in the literatures (9-11, 42, 43, 53-58).Proinflammatory macrophages are known to upregulate their

Proinflammatory role of CTRP6
primary glucose transporter (GLUT1) leading to increased glucose uptake (10).Despite the presence of oxygen, upon proinflammatory activation, macrophages shift to rely on glycolysis rather than on mitochondrial oxidative metabolism to fuel their energetic needs (44,59,60).This "Warburg-like" metabolism with elevated lactate secretion (61) has been extensively documented in M1-polarized macrophages (42,60).While glucose flux through glycolysis and the pentose phosphate pathway are greatly elevated in proinflammatory macrophages, the mitochondrial oxidative metabolism is correspondingly downregulated (9-11, 42, 43, 53-58).The mechanisms underpinning the "Warburg-like" metabolism in proinflammatory macrophages are being actively investigated (44,59,60).Recent studies have suggested that the tricarboxylic acid cycle within mitochondria that supplies the reducing equivalents (NADH2 and FADH2) to the electron transport chain is broken at two places, leading to elevated citrate and succinate (42,44).Succinate stabilizes hypoxiainducible factor 1-alpha, leading to the upregulated expression of glycolytic enzymes and glucose transporters (11).Increased succinate oxidation by succinate dehydrogenase (part of complex II) without the corresponding increase in ATP synthesis results in reverse electron transport within the electron transport chain that enhances mitochondrial ROS production (48).These mechanisms are also likely responsible for the observed increase in glycolytic rate, lactate secretion, and the corresponding reduction in oxidative metabolism in BMDMs treated with CTRP6.
CTRP6 belongs to the C1q family with over 30 members, united by a shared globular C1q domain (62,63).Interestingly, several members of this family have also been demonstrated to play a role in regulating macrophage function and polarization.For example, complement C1q (64-66), adiponectin (67-69), CTRP4 (70), and CTRP9 (71) have been shown to polarize macrophages toward the antiinflammatory M2 phenotype.In contrast, CTRP1 appears to polarize macrophages toward the proinflammatory phenotype (72).Depending on the context, CTRP3 has been shown to polarize macrophages toward either the proinflammatory or antiinflammatory phenotype (73,74).Thus, in a broader context, our current findings along with previous studies by others suggest the C1q family to be an important class of secreted regulators of macrophage function across different physiological and disease states.
Two limitations of the study are noted.First, the dose of CTRP6 we used in our in vitro studies was empirically determined.We do not know the concentration of CTRP6 in the adipose tissue microenvironment, and the lack of a validated ELISA specific for mouse CTRP6 precluded such measurement.Given CTRP6 is an autocrine and paracrine factor markedly upregulated in the fat compartment in obesity, the local concentration of CTRP6 that adipose tissue macrophages potentially exposed to could be high and thus the dose we used is likely within the physiological range encountered in vivo.Importantly, our loss-of-function studies in BMDMs and in mice corroborated our gain-of-function studies using recombinant CTRP6.Second, we do not know the receptor on macrophages that mediates the biological action of CTRP6.
While it has been shown that CTRP6 can bind to soluble complement C3, collectin-11, and certain sugar such as I-fucose (75,76), no plasma membrane receptor on macrophages has been identified that binds CTRP6.Since CTRP6 alone can rapidly and directly activate BMDMs, we presumed that a CTRP6-specific receptor is expressed by macrophages, the identity of which remains to be discovered.
In summary, our data highlighted the direct mechanistic action of CTRP6 on promoting macrophage proinflammatory response.Since mice lacking CTRP6 have reduced LPSinduced systemic inflammation, as well as decreased adipose tissue inflammation in obesity (25), approaches to neutralize the activity of CTRP6 in pathological settings may prove beneficial.

Animals
C57BL/6J WT mice were purchased from The Jackson Laboratory.The Ctrp6 whole-body KO mice (129S5-C1qtnf6 tm1Lex /Mmucd; stock number 031616-UCD) were obtained from Mutant Mouse Regional Resource Center, and backcrossed for >10 generations onto a C57BL/6J genetic background (25).Heterozygous mice were intercrossed to obtain WT (+/+) and Ctrp6 KO (−/−) mice.All mice were housed in polycarbonate cages under a 12-h/12-h light/dark photocycle and fed a standard chow (9% fat; Lab Diet) with free access to water.All mouse protocols were approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University School of Medicine.All animal experiments were conducted in accordance with the National Institute of Health guidelines and followed the standards established by the Animal Welfare Acts.

Recombinant mouse CTRP6 production
Recombinant full-length mouse CTRP6 containing a C-terminal FLAG epitope tag (DYKDDDDK) was produced in suspension FreeStyle 293-F cells (Thermo Fisher Scientific) at the Mammalian Cell Expression Core of The Johns Hopkins University School of Medicine.For 1 l expression, 1 mg of purified CTRP6 expression plasmid (in pCDNA3.1 vector) were transfected with PEI (Polysciences, Inc) at a ratio of 1:3 into FreeStyle 293-F cells at a concentration of 2 × 10 6 to 2.4 × 10 6 cells per ml and incubated at 37 C incubator for 3 days.Cells were cultured in FreeStyle 293 expression medium (Thermo Fisher Scientific).The 1 l medium was harvested via centrifugation, sterile filtered, and subjected to affinity chromatography using an anti-FLAG M2 affinity gel (Sigma-Aldrich, A2220) according to the manufacturer's protocol.Purified protein was dialyzed against 20 mM Hepes (pH 8.0) containing 135 mM NaCl using a 10-kDa molecular weight cutoff dialysis cassette (Thermo Fisher Scientific, 66810) and concentrated with a 10-kDa cutoff Amicon Ultra-15 centrifugal filter unit (Millipore, UFC901024).Protein concentration was determined using the bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific, 23227) and samples were aliquoted and stored at −80 C.

Proinflammatory role of CTRP6 Macrophage cell culture
Primary mouse BMDMs were cultured as previously described (25).Briefly, BMDMs were isolated from the femurs and tibias of C57BL/6J WT mice or Ctrp6 KO mice between 8 and 12 weeks of age and cultured on Petri dishes in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% Hepes, 1% sodium pyruvate plus 10 ng/ml recombinant macrophage colony-stimulating factor (M-CSF; R&D Systems, 416-ML-010).Differentiated cells were refed on day 3. On day 7, differentiated BMDMs were lifted by trypsinization, collected, counted, and replated in complete RPMI 1640 medium in culture plates.After an overnight incubation, BMDMs were treated accordingly.The purity of BMDMs was assessed by flow cytometry.Approximately 80% of the cells were positive for F4/80 and Cd11b (Fig. S2).For CTRP6 treatment, cells were treated with vehicle control (Hepes) or purified recombinant CTRP6 in RPMI 1640 with 0.2% bovine serum album for varying periods of time.For pretreatment experiments, BMDMs were pretreated with 5 μg/ ml CTRP6 for 1 h, followed by stimulation with 100 ng/ml LPS (Sigma-Aldrich Escherichia coli 055: B5, L2880) in the presence of CTRP6 for varying periods of time.For cotreatment experiments, BMDMs were treated with 5 μg/ml CTRP6 in the presence of 100 ng/ml LPS for 6 h.In experiments involving pharmacologic inhibitors, BMDMs were pretreated for 1 h with dimethyl sulfoxide (vehicle control) or p38-MAPK inhibitor SB203580 (10 μM, Selleckchem, S1076), MEK/Erk1/2 inhibitor PB98059 (20 μM, Tocris Bioscience, 1213), or NF-κB inhibitor Bay 11-7082 (Bay11, 5 μM, Selleckchem, S2913), and subsequently treated with 5 μg/ml CTRP6 or vehicle control (Hepes) for an additional 24 h in the presence of each inhibitor.For WT and Ctrp6-KO BMDMs, cells were stimulated with 100 ng/ml LPS for varying periods of time as indicated.After treatment, cells were washed and harvested for immunoblotting and quantitative real-time PCR (qRT-PCR).Supernatants were collected to measure cytokine production by ELISA.
Primary AMs were isolated from the bronchoalveolar lavage (BAL) fluid of C57BL/6J WT mice or Ctrp6-KO mice as previously described (77).Briefly, the thoracic cavity of anesthetized mice was opened to expose the airway, then BAL fluid was collected by cannulating the trachea and lavaging the lung using prechilled PBS with 1 mM EDTA.Multiple flushes of BAL fluid were pooled and centrifuged at 250g at 4 C for 10 min.The pelleted AMs were resuspended and cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 1% penicillin/streptomycin, 10 mM Hepes plus 25 ng/ml M-CSF.After 24 h, the adherent AMs were stimulated with 200 ng/ml LPS or 20 ng/ml IL-4 for 24 h for the M1 macrophage polarization and M2 macrophage polarization, respectively.
Kupffer cells were isolated from the liver of Ctrp6-KO mice by a modified in situ perfusion procedure as previously described (78).Briefly, the inferior vena cava was cannulated and the liver was perfused with liver perfusion media (Thermo Fisher Scientific, 17701) followed by 0.1% collagenase IV digest (Thermo Fisher Scientific, 17104019).The disassociated liver was then minced by spatula, filtered through a 100 μM strainer, and centrifuged at 100g for 3 min.The supernatant enriched in Kupffer cells was loaded onto a Percoll gradient (GE Health-Care, 17-0891-01) of 25% and 50% for density gradient centrifugation at 2300g for 30 min at 4 C without a break.The interphase ring was collected, resuspended, and cultured in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/ streptomycin.After 2 h, nonadherent cells were removed by changing the culture medium.The adherent fraction was washed and stimulated with 100 ng/ml LPS for 24 h.

Flow cytometry
Day-6 differentiated BMDMs were collected and washed, then incubated in fluorescence activated cell sorting (FACS) buffer (0.5% BSA and 2 mM EDTA in PBS) with Fc-block (CD16/32) (BioLegend, 101319) on ice for 10 min to reduce nonspecific binding.Cells were stained with F4/80 and CD11b on ice for 30 min in the dark.After multiple washes to remove excess antibodies, cells were assayed on an Attune N × T Acoustic Focusing Cytometer (Invitrogen) and data were analyzed using FlowJo software v.10.8.0 (Tree Star Inc).The antibody information is provided in Table S6.

Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from liver tissues or cells using TRIzol reagent (Invitrogen, 15596018) according to the manufacturer's protocol.The first-strand complementary DNAs were reverse transcribed using iScript cDNA Synthesis Kit (Bio-Rad, 1708891).qRT-PCR was performed using the Universal SYBR Green Supermix (Bio-Rad, 1725124) on a CFX Connect system (Bio-Rad).Results were normalized to 36B4 and expressed as fold changes using the 2 −△△CT method (79).The sequences of all primers are listed in Table S5.

Immunoblotting
Proteins from tissues or cells were extracted in RIPA buffer (Sigma-Aldrich, R0278) freshly supplemented with protease inhibitor cocktails (Roche, 11836170001) and phosphatase inhibitor cocktails (Roche, PhosSTOP, 04906837001).Protein concentrations were quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23227).Equal amounts of total protein lysates were analyzed by standard Western blot procedures using either the Mini-Protean tris-glycine extended gels or the Criterion tris-glycine extended Midi Protein gels (Bio-Rad).Protein bands were developed using the ECL substrate (Millipore, WBLUC0500) and visualized by FluorChem Q Imager (Alpha Innotech) followed by densitometry quantification using the ImageJ software (80).Antibody information is provided in Table S6.

Cytokine measurements
Mouse IL-6 and MCP-1 in cell culture supernatants were measured by commercially available ELISA kit (R&D Systems, M6000B and MJE00B) according to the manufacturer's instructions.For quantifying TNF-α level in cell culture supernatants and mouse serum, mouse TNF-α ELSIA kit (R&D Systems MTA00B) was used according to the manufacturer's instructions.

Lactate assay
The levels of lactate in cell culture supernatants were measured with the Lactate Assay Kit (Abcam, ab65330) according to the manufacturer's instructions.

Mitochondrial ROS measurement
For measuring mitochondrial ROS generation, BMDMs were treated with 5 μg/ml CTRP6 with or without 100 ng/ml LPS for 6 h, followed by incubation with 5 μM MitoSOX (Thermo Fisher Scientific, M36008) in Hanks' balanced salt solution plus calcium and magnesium for 15 min at 37 C, protected from light.After staining, cells were washed and counterstained with Hoechst 33258 (Sigma-Aldrich, 94403) and imaged using a fluorescence microscope.The positive cells were counted and normalized to total cell number.

Seahorse respirometry assay
Oxygen consumption rate and ECAR were measured using an XF96 Extracellular Flux Analyzer (Seahorse Bioscience).Briefly, BMDMs were seeded at 8 × 10 4 cells per well in Seahorse XF96 tissue culture plates (Agilent Technologies) and cultured overnight before treated with 5 μg/ml CTRP6 with or without 100 ng/ml LPS for 6 h.The ECAR in BMDMs were measured under basal conditions and following the addition of 20 mM glucose.Changes in oxygen consumption rate were detected under basal conditions and following the sequential addition of 2.5 μM oligomycin, 2 μM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone and 0.5 μM antimycin A/ rotenone (all the chemical compounds were from Agilent Technologies).Results were collected with Wave software version 2.6 (Agilent Technologies).Data were normalized to the absorbance at 450 nm after incubation with watersoluble tetrazolium salts cell proliferation reagent (Roche, 05015944001) as a measure of cell number in each well.

LPS-induced inflammation and hypothermia
For endotoxemia model, 8 to 12-week-old male Ctrp6 KO mice and age-matched WT littermates were administered with LPS (E. coli 055: B5, L2880, Sigma-Aldrich) at 5 mg/kg by intraperitoneal injection.The mice had normal access to food and water and the deep colonic temperature was monitored with a digital thermocouple thermometer (BAT-12, Physitemp Instruments) before and 6 h after LPS injection.Blood was collected before and at 2 and 6 h after LPS injection, placed at room temperature (RT) for 2 h and then centrifuged for 5 min at 10,000g.Serum samples were collected and stored at −80 C. Mice were euthanized at 6 h after LPS injection and tissues were snap-frozen and stored at −80 C.

RNA sequencing and analysis
Bulk RNA sequencing was performed on total RNA isolated from BDMDs treated with 5 μg/ml CTRP6 or vehicle control (Hepes) for 6 h.There were four biological replicates for each group.Library was prepared and sequenced at Novogene on an Illumina NovaSeq 6000 platform with total raw reads of 20 million per sample.The RNA-seq base call files were converted to fastq files by using the bcl2fastq (version 2.15.0.4) program.RNA-seq reads were then aligned to the Ensembl release with STAR version 2.0.4b.Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread feature count version 1.4.5.Quantification, normalization, and DEGs were determined with the Cufflinks package (version 2.2.1).DEGs were used as an input for pathway analysis through Ingenuity Pathway Analysis suite (www.ingenuity.com).The R/Bioconductor package heatmap3 was used to display heatmaps or annotated KEGG graphs across groups of samples for each KEGG pathway with a Benjamini-Hochberg false-discovery rate adjusted p-value less than or equal to 0.05.DEGs were defined as ones with at least 0.5 fragments per kilobase of transcript per million mapped reads level of expression in at least 1 of the conditions and a Q-value <0.05.The RNA-seq data was deposited in the NCBI database, with the Sequence Read Archive (SRA) accession # PRJNA947594.

Reagents for proteomics analysis
Titanspheres (TiO2, 5 μm beads) were from GL Sciences Inc. L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) treated trypsin was from Worthington Biochemical Corp.All other reagents used in this study were from Thermo Fisher Scientific.
Cell lysis, protein digestion, and TMT labeling BMDM were treated with 5 μg/ml CTRP6 or vehicle control (Hepes) for 30 min.The treatments were carried out in triplicates.Cells were washed with ice-cold PBS, collected and lysed in lysis buffer (8 M urea, 20 mM Hepes, pH 8.0, 2.5 mM sodium pyrophosphate, 1 mM disodium β-glycerophosphate, 1 mM sodium orthovanadate, and 10 mM sodium fluoride) by sonication.After centrifugation at 17,000g at 15 C for 20 min, the protein lysates were collected and the protein concentration was determined using BCA assay (Pierce).An equal amount of protein from each sample was reduced by DTT at a final concentration of 5 mM at 60 C for 20 min and alkylated using 10 mM iodoacetamide for 20 min at RT in the dark.For tryptic digestion, protein extracts were diluted in 20 mM Hepes pH 8.0 to a final concentration of 2 M urea and incubated with TPCK-treated trypsin at 25 C overnight.Protein digests were acidified by 1% TFA and subjected to centrifugation at 2000g at RT for 5 min.The supernatant of protein digests was loaded onto a Sep-Pak C18 column (Waters, WAT051910) equilibrated with 0.1% TFA.Columns were washed with 12 ml of 0.1% TFA and peptides were eluted in 6 ml of 40% acetonitrile (ACN) with 0.1% TFA.Eluted peptides were lyophilized and subjected to tandem mass tag (TMT) labeling.TMT labeling was carried out according to the manufacturer's instructions.Briefly, 250 μg of tryptic peptides from each sample was reconstituted in 100 μl of 50 mM Proinflammatory role of CTRP6 triethylammonium bicarbonate buffer (TEABC) buffer and mixed with a 6-plex TMT reagent reconstituted in 41 μl of anhydrous ACN and incubated at RT for 1 h.All the labeled peptides from each sample were equally mixed, dried completely in a vacuum concentrator, and kept at −80 C.

Fractionation of peptides by basic reversed-phase liquid chromatography
TMT-labeled peptide mixtures were resuspended in 1 ml of 10 mM TEABC, pH 8.0 and loaded on a XBridge BEH C18 Column, 130 Å, 5 μm, 4.6 mm × 250 mm (Waters, Cat # 186003117), and fractionated on an Agilent 1100 Series HPLC system by basic reversed-phase chromatography at a flow rate of 400 μl/min.Mobile phase consisted of 10 mM TEABC, pH 8.0 (buffer A) and 10 mM TEABC, 90% ACN, pH 8.0 (buffer B).After loading 1 ml of sample (6.4 mg) onto the column, the peptides were separated using the following gradient: 5 min isocratic hold at 2% B, 5 to 7% solvent B in 2 min; 5 to 40% solvent B in 63 min; 40 to 100% solvent B in 2 min; hold at 100% solvent B for 3 min, 100 to 2% solvent B in 1 min, hold at 2% solvent B for 4 min for a total gradient time of 80 min.Using 96 × 1 ml well plates (Thermo Fisher Scientific, #7701-5200), fractions were collected for a total of 96 fractions through the elution profile of the separation.A total of 5% of collection from each well were merged into 12 fractions and dried by vacuum centrifugation for the liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the proteomic changes in cells.The rest of the collection from each well were merged into 12 fractions and dried by vacuum centrifugation for TiO 2 -based phosphopeptide enrichment.

Mass spectrometry data analysis
The tandem mass spectra were searched using the Andromeda search algorithm SEQUEST HT (83) against a mouse UniProt database (released on May 2018) embedded in the Proteome Discoverer platform (Thermo Fisher Scientific, version 2.2).The search parameters were set as follows: a maximum of two missed cleavages, carbamidomethylation at cysteine and TMT at lysine and peptide N terminus as a fixed modification and oxidation at methionine, acetylation at protein N terminus, and phosphorylation at serine, threonine, and tyrosine as variable modifications.The mass tolerances for MS and MS/MS were set to 10 ppm and 0.02 Da, respectively.The reverse type of the target-decoy analysis was chosen.False discovery rates for both peptide and protein level filterings were set to 0.01.PhosphoRS (84) was used to calculate phosphorylation site probability.The minimum peptide length was set to six amino acids.The minimum number of peptides for protein identification was set to 1.The TMT reporter ion intensities were used to calculate the abundance changes of proteins and phosphopeptides in CTRP6versus control vehicle-treated BMDMs.

Statistical analysis
All results are expressed as mean ± standard error of the mean (SEM).Statistical analysis was performed with Prism 9 software (GraphPad).Data were analyzed with two-tailed Student's t-tests or by One-way ANOVA.Values were considered to be significant at p <0.05.

Data availability
All data described in this manuscript is contained in the main text or Supplementary materials.RNA-seq data has been deposited in NCBI database, with the Sequence Read Archive (SRA) accession # PRJNA947594.The mass spectrometry proteomics data have been deposited to the ProteomeXchange

Figure 1 .
Figure 1.CTRP6 promotes a macrophage proinflammatory phenotype.A, a volcano plot showing significantly upregulated and downregulated genes in BMDMs treated with CTRP6 or vehicle control.B, the top upregulated pathways in CTRP6-treated BMDMs, as indicated by the KEGG pathway analysis.C, a heatmap showing some of the differentially expressed proinflammatory and antiinflammatory genes obtained from RNA-seq in CTRP6-treated BMDMs.Color gradient reflects row Z-score values.D, relative mRNA expression of proinflammatory and antiinflammatory genes in BMDMs treated with CTRP6 for 6 h (n = 6).Data are shown as the mean ± SEM.Representative of three independent experiments.One-way ANOVA followed by Tukey's post hoc test.*p < 0.05; **p < 0.01.E, a heatmap showing differentially expressed genes (from RNA-seq) involved in MAPK and NF-κB signaling pathways in CTRP6-treated BMDMs.Color gradient reflects row Z-score values.BMDM, bone marrow-derived macrophages; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK, mitogen-activated protein kinase.

Figure 2 .
Figure 2. Phosphoproteomic analysis reveals enrichment for MAPK signaling induced by CTRP6 in macrophages.A, schematic illustration of TMT 6 plex-based workflow for quantitative phosphoproteomic analysis [see details in Experimental procedures].B, a volcano plot showing all the phosphopeptides that are upregulated or downregulated by CTRP6-treated BMDMs.Fold-change cutoff was placed on ±1.2-fold, as explained in Result section.C, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (top) and Gene Ontology biological process analysis (bottom) for phosphoproteins with significantly altered phosphorylation level.BMDM, bone marrow-derived macrophage; MAPK, Mitogen-activated protein kinase; TMT, tandem mass tag.

Figure 3 .
Figure 3. CTRP6 activates the MAPK and NF-κB signaling pathways to promote a proinflammatory response in macrophages.A, Western blot analysis of MAPK and NF-κB signaling pathways activated by CTRP6 in BMDMs over a 180-min time course.Phospho (P) and total Erk1/2, p38-MAPK, JNK, IκB, p65 subunit of NF-κB are indicated.The band densities of phospho-Erk1/2, p38-MAPK, JNK, and p65 were first normalized to their corresponding total protein.In the case of IκB, it was normalized to GAPDH.The relative band density shown at 30, 60, and 180 min was normalized to the band density at time 0 (which was set to 1).Data are shown as the mean ± SEM.Representative of two independent experiments.One-way ANOVA followed by Tukey's post hoc test.**p < 0.01.B, secretion of cytokines from BMDMs pretreated with p38 MAPK inhibitor SB203580, MEK/Erk1/2 inhibitor PD98059, and NF-κB inhibitor Bay 11-7082 for 1 h, followed by cotreatment with CTRP6 for 24 h (n = 10).Data are shown as the mean ± SEM.Representative of three independent experiments.Two-tailed unpaired Student's t test.**p < 0.01; ****p < 0.0001.BMDM, bone marrow-derived macrophage; MAPK, mitogen-activated protein kinase.JNK, c-Jun N-terminal kinase.

Figure 4 .
Figure 4. CTRP6 augments LPS-stimulated inflammatory response in macrophages.A, Ctrp6 expression in BMDMs stimulated with LPS (100 ng/ml) for 0 to 24 h (n = 6).Data are shown as the mean ± SEM.Representative of two independent experiments.One-way ANOVA followed by Tukey's post hoc test.**p < 0.01.BMDMs from Ctrp6 KO mice were used as a negative control.B, Western blot analysis of Ctrp6 in BMDMs stimulated with LPS (100 ng/ml) for 0 to 24 h (top panel).Band intensities were quantified and shown on the bottom panel (n = 4).Data are shown as the mean ± SEM.Representative of two independent experiments.One-way ANOVA followed by Tukey's post hoc test.*p < 0.05, **p < 0.01.C-E, secretion of TNF-α (C), IL-6 (D), and MCP-1 (E) from BMDMs pretreated with CTRP6 (5 μg/ml) for 1 h, followed by LPS (100 ng/ml) stimulation for 0 to 24 h in the presence of CTRP6.Data are shown as the mean ± SEM.Representative of two independent experiments with five samples per group.Two-tailed unpaired Student's t test.*p < 0.05, **p < 0.01.F, expression of proinflammatory genes in BMDMs pretreated with CTRP6 for 1 h, followed by LPS stimulation for 24 h in the presence of CTRP6 (n = 6).Data are shown as the mean ± SEM.Representative of three independent experiments.Two-tailed unpaired Student's t test.*p < 0.05.G, Western blot analysis of MAPK and NF-κB signaling pathways activated in BMDMs by LPS alone, or LPS in combination with CTRP6 (pretreatment for 1 h and thereafter).Phospho (P) and total Erk1/2, p38-MAPK, IκB, p65 subunit of NF-κB are indicated.For the phospho-Erk1/2 and phospho-p-38 blots, original sample loading was in reverse order.For consistency, we switched the group such that vehicle-treated samples came before the CTRP6-treated samples.We inserted a line on the gel images to indicate where splicing occurred.H, the band densities of phospho-Erk1/2, p38-MAPK and p65 were first normalized to their corresponding total protein.In the case of IκB, it was normalized to β-actin.The relative band density shown at 15, 30, 60, 180, and 360 min was normalized to the band density at time 0 (which was set to 1).Data are shown as the mean ± SEM.Representative of two independent experiments with two samples per group.Two-tailed unpaired Student's t test.*p < 0.05, **p < 0.01.BMDM, bone marrow-derived macrophage; IL, interleukin; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemoattractant protein-1; TNF, tumor necrosis factor.

Figure 6 .
Figure 6.CTRP6-deficient BMDMs have reduced LPS-induced inflammation gene expression and signaling.A and B, expression of proinflammatory and antiinflammatory genes in WT and Ctrp6 KO BMDMs at baseline (A), and after LPS treatment (B) for 24 h (n = 6).Data are shown as the mean ± SEM.Representative of three independent experiments.Two-tailed unpaired Student's t test.*p < 0.05, **p < 0.01.C, Western blot analysis of MAPK and NF-κB signaling pathways activated before (time 0) and after LPS stimulation in WT and CTRP6-deficient BMDMs.Phospho (P) and total Erk1/2, p38-MAPK, IκB, p65 subunit of NF-κB are indicated.D, the band densities of phospho-Erk1/2, p38-MAPK and p65 were first normalized to their corresponding total protein.In the case of IκB, it was normalized to β-actin.The relative band density shown at 15, 30, 60, 180, and 360 min was normalized to the band density at time 0 (which was set to 1).Data are shown as the mean ± SEM.Representative of two independent experiments with two samples per group.Two-tailed unpaired Student's t test.*p < 0.05, **p < 0.01.BMDM, bone marrow-derived macrophage; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase.

Figure 7 .
Figure 7. Mice lacking CTRP6 have attenuated LPS-induced inflammation and hypothermia.A, secretion of TNF-α, IL-6, and MCP-1 from WT and Ctrp6 KO BMDMs treated with LPS for 0 to 24 h.Data are shown as the mean ± SEM.Representative of two independent experiments with five samples per group.Two-tailed unpaired Student's t test.*p < 0.05, **p < 0.01.B, serum TNF-α levels in WT and Ctrp6 KO mice after intraperitoneal LPS injection at a dose of 5 mg/kg.C, changes in body (rectal) temperature before and after LPS injection (n = 8 per group).D, expression of proinflammatory genes in the liver of WT and Ctrp6 KO mice at 6 h after LPS injection (n = 5 per group).Data are shown as the mean ± SEM.Representative of three independent experiments.Twotailed unpaired Student's t test.*p < 0.05, **p < 0.01.IL, interleukin; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein-1; TNF, tumor necrosis factor.

Table 2 A
partial list of CTRP6-downregulated phosphoproteins in BMDMs