The Lcn10 global knockout mouse model in C57Bl/6 background was initially purchased from KOMP Repository at UC Davis (Stock # 048394-UCD). Wild-type (WT) C57BL/6 mice and CD45.1 mice (B6.SJL-Ptprca Pepcb/BoyJ) were purchased from Jackson Laboratory. Mice were housed and bred in specific-pathogen-free and temperature-controlled conditions, with 12-h light-dark cycles at the University of Cincinnati Animal Care Facility. All animal procedures followed the criteria of Care and Use of Laboratory Animals by the National Institutes of Health and approved by the University of Cincinnati Animal Care and Use Committee. To induce Type 2 diabetes, male mice (4–5 weeks old) were initially fed with a high-fat diet (HFD, Cat. # D12492, Research Diet, New Brunswick, NJ) for four weeks, followed by intraperitoneally injected with a single dose of streptozotocin (STZ, 100 µg/g body weight; Sigma-Aldrich, Cat. # S0130). Then, these mice were maintained on HFD feeding throughout the study. The onset of diabetes will be determined by a blood glucose level higher than 250 mg/dl at 48 h after STZ injection. Non-diabetic (ND) control mice were fed with a standard chow diet and received the same volume of citrate buffer injection.

Mouse L-929 cell line was initially purchased from ATCC (CCL-1) and maintained in DMEM medium supplemented with 10% FBS, 1% penicillin/streptomycin solution, and 10 mM HEPES buffer at 37°C with 5% CO2, and 95% relative humidity. L-929 cell culture medium was collected after ten days of culture and centrifuged at 500g for 10 min, then passed through a sterile 0.45 µM filter (Millipore, Cat. # S2HVU02RE). Next, the supernatants (enriched in monocyte-colony stimulating factor, M-CSF) were aliquoted in 50 ml tubes and stored at -80 C° until use. Bone marrow-derived macrophages (BMDMs) were prepared as previously described (32, 33). Briefly, WT and Lcn10-KO mice were terminally anesthetized, and bone marrows from femur and tibia bones were flushed out using cold PBS containing 2% FBS. The marrows were filtered through a 70 µm cell strainer, followed by the removal of red blood cells (RBC) in ammonium-chloride-potassium lysis buffer (BioLegend, Cat. # 420302) for 5 min at room temperature (RT). Next, cells were grown in BMDMs complete medium (DMEM supplemented with 10% FBS, 15% L929 cell culture supernatant, 1% penicillin/streptomycin solution, 10mM HEPES buffer) and allowed to differentiate for seven days. On day 3, an additional 10 ml BMDMs complete medium was added to the culture dish. For BMDMs phenotypic polarization, cells were plated in 6-well plates with BMDMs complete medium overnight and then treated with LPS (Sigma, Cat. # L4391), IFN-γ (R&D, Cat. # 485-MI-100), Palmitate (Sigma, Cat. # P9767), oxidized Low-Density Lipoprotein (oxLDL, Invitrogen, Cat. # L34357) or IL-4 (R&D, Cat. # 404-ML-010) at the indicated doses and time points. For Nr4a1 agonist treatment, BMDMs from WT and Lcn10 KO mice were pretreated with Cytosporone B (CsnB, 5 µM) for 30 minutes, followed by LPS (10 ng/ml) + IFN-γ (10 ng/ml) stimulation at indicated time point.

qRT-PCR was conducted as previously described (34). In brief, RNAs from BMDMs or heart macrophages were extracted using miRNeasy Mini Kit (Qiagen, Cat. # 217004). According to the manufacturer’s instructions, 0.5 to 1 µg RNA was converted to complementary DNA (cDNA) using Superscript II Reverse Transcriptase (Invitrogen, Cat. # 18064014). Then the obtained cDNA products were mixed with SYBR Green Hi-ROX Master Mix (Radiant, Cat. # QS2050) and performed in triplicate using the ABI StepOnePlus Real-Time PCR System. Relative mRNA levels were normalized to GAPDH as an internal control for each sample and calculated using the delta-delta CT method (2^-ΔΔCt). The primer sequences used for amplification are listed in Supplementary Table S1 .

To analyze BMDMs, cells were first incubated with CD16/32 Ab (clone 93) (eBioScience, Cat. # 14-0161-81, 1:100 dilution) to block the nonspecific binding to Fc receptors. After washing twice with FACS buffer (1XPBS without Calcium and Magnesium, 1% BSA, 1 mM EDTA), cells were stained with fluorophore-coupled antibodies for 30 minutes at 4°C. The antibodies were listed in Supplementary Table S2 . For analysis of heart macrophages, methods were adopted and modified as previously described (35, 36). In brief, mice were anesthetized with ketamine (90 mg/kg BW) and xylazine (10 mg/kg BW), followed by perfusing with 15 ml of cold PBS via the left ventricle. The heart was thoroughly minced and digested in HBSS with 1 mg/mL Collagenase I (Worthington, Cat. # LS004196), 1 mg/mL Collagenase II (Worthington, Cat. # LS004177), 1 mg/mL Dispase II (Sigma, Cat. # D4693). After 45 minutes of incubation at 37°C with gentle agitation, the digested heart pieces were passed through a 40 µm cell filter to obtain a single-cell suspension, followed by centrifugation at 500g for 5 minutes at 4°C. The resulting cell pellets were resuspended in 1 ml RBC lysis buffer (BioLegend, Cat. # 420302), incubated for 3 minutes at room temperature, and washed with PBS. Following this, samples are blocked with CD16/32 Ab and stained for surface markers. The antibodies were listed in Supplementary Table S2 . Flow cytometry was performed on a BD LSRFortessa analyzer (Research Flow Cytometry Core, Cincinnati Children’s Hospital Medical Center). The obtained data were analyzed using FCS Express V7 Software (De Novo, USA).

Heart macrophages were isolated using the MagniSort™ Mouse F4/80 Positive Selection Kit (Invitrogen, Cat. # 8802-6863) according to the manual. Briefly, a single-cell suspension of the heart was prepared as described above. Then the cells were incubated with biotinylated F4/80 selection antibodies for 10 minutes at RT, followed by centrifugation at 300g for 5 minutes in a 12 X 75 mm, 5 ml tube. The cell pellets were resuspended in cell separation buffer and incubated with magnetic beads for 10 minutes at RT. The tube containing samples was placed in the magnet for 5 minutes, and the supernatant was discarded. After total three times of positive selections in the magnet and washing with cell separation buffer, cells were collected by centrifugation and ready for further experiments.

Bone marrow transplantation was performed with slight modifications (37, 38). CD45.1 male recipient mice (8-10 weeks old) were lethally irradiated with 1000 cGy (split into two doses, 500 cGy/dose, 4 hours apart) in a XenX cabinet X-ray irradiator (Preclinical Imaging Core, University of Cincinnati). Within 24 hours after irradiation, all recipient mice were retro-orbitally injected with freshly isolated bone marrow cells (~ 5 x 10⁶ cells in 150 ul of 2% FBS in PBS) from CD45.2 donor mice (WT and Lcn10-KO male mice, 4-6 weeks old). The bone marrow cells were prepared as described earlier in section 2.2 without RBC lysis. After a 2-week recovery period, during which mice were administered a chow diet (CD), mice were then fed with an HFD for 4 weeks, followed by a single dose of streptozotocin injection. Then these mice were maintained on the HFD-feeding paradigm for the indicated times until sacrifice. All recipient mice were given free access to water supplemented with 0.25 mg/ml enrofloxacin (Alfa Aesar, Cat. # J60023) one week before and four weeks after irradiation.

For BMDMs staining, cells seeded on the coverslip were stimulated with LPS (10 ng/ml) + IFN-γ (10 ng/ml) for 1 hour. After washing with PBS two times, cells were fixed with 4% PFA for 20 minutes, followed by permeabilization in 0.3% Triton X-100 in PBS for 15 minutes at RT. Then cells were blocked with 2% BSA in PBS for 1 hour at RT. Next, samples were stained with primary Nr4a1 antibody at a dilution of 1:50 (Affinity, Cat. # DF7850) overnight at 4°C. After washing, cells were incubated with Alexa Fluor Plus 488 secondary antibody at a dilution of 1:200 (Invitrogen, Cat. # A32723) for 1 hour at RT. Finally, samples were mounted with ProLong Diamond Antifade Mounting medium with DAPI (Invitrogen, Cat. # P36962). Images were acquired on Zeiss LSM710 LIVE Duo Confocal Microscope (Live Microscopy Core, University of Cincinnati).

Cardiac function was evaluated in vivo by trans-thoracic echocardiography using Vevo 2100 ultrasound imaging system (VisualSonics, FUJIFILM, Toronto, Canada) with a 40-MHz linear array transducer as previously described (39). Left ventricle (LV) end-systolic inner diameters (LVIDs), LV end-diastolic inner diameter (LVIDd), ejection fraction (EF), and fractional shortening (FS) were analyzed using Vevo 2100 analysis system with a cardiac package, and all measurements were repeated at least three times.

For glucose tolerance tests, mice were fasted overnight and administered a 45% glucose solution (0.5 g/kg) intraperitoneally. For insulin tolerance tests, mice were fasted for 6 hours and then intraperitoneally injected with insulin (0.75 U/kg). Blood samples were collected from the tail vein at the indicated time points over a 2-hour period. Blood glucose levels were measured using a Contour Next EZ glucometer combined with Contour Next blood glucose test strips.

All data were analyzed with GraphPad Prism version 8.4 and presented as the mean ± SEM otherwise specified. A two-tailed Student’s t-test was performed when two groups were compared. Differences between more than two groups were determined by one-way or two-way ANOVA. A p value < 0.05 was considered statistically significant.

To testify whether Lcn10 contributes to macrophage function, we first characterized the gene expression of Lcn10 in macrophages in response to multiple stimuli. To mimic T2D conditions in vivo, BMDMs were treated with LPS + IFNγ (pro-inflammatory inducer), palmitate (a saturated fatty acid), and oxLDL (oxidized lipids) for 12 hours. We observed that the expression of Lcn10 was downregulated by 80%, 60%, and 20%, respectively ( Figures 1A–C ), compared to controls. We also detected the expression levels of Lcn2, another lipocalin family member that is well-characterized for its role in regulating macrophage function (40, 41). Consistent with previous reports (42), the mRNA levels of Lcn2 expression in macrophages were dramatically increased by 6800-fold and 35-fold when challenged with LPS + IFNγ or palmitate, respectively ( Figures 1E, F ); while no significant change in Lcn2 expression was observed in the oxLDL-treated group compared to the control group ( Figure 1G ). Similar results were observed in macrophages isolated from the hearts of T2D mice ( Figures 1D, H ) compared to those from chow-fed non-diabetic (ND) mice. Collectively, these results indicate that Lcn10 may play a critical role in regulating macrophage function upon metabolic challenge.

We next went on to evaluate the functional role of Lcn10 in the regulation of macrophage polarization under various metabolic stresses. Given that lipocalin featured as a secreted protein (42, 43) and Lcn10 was downregulated as shown above, a global Lcn10-knockout (KO) mouse model was used in our study ( Figures 2A, B ). First, we isolated BMDMs from WT and Lcn10-KO mice and cultured them for 7 days in vitro. As shown in Figures 2C, D , there was no difference in morphology and differentiation of BMDMs from WT and KO mice. We then treated WT and Lcn10-KO BMDMs with LPS + IFNγ and determined the gene expression of pro-inflammatory markers using qRT-PCR. Notably, loss of Lcn10 did not alter the basal expression levels of these genes ( Figures 2E–H and Supplementary Figures S1A–F, S2A–I ). However, when treated with LPS + IFNγ, BMDMs from Lcn10-KO groups exhibited significantly higher expression of pro-inflammatory marker genes, including iNOS ( Figure 2E ), IL-6 ( Figure 2F ), IL-1β ( Figure 2G ), TNFα ( Figure 2H ), and other pro-inflammatory genes (i.e., CXCL1, CXCL9, CXCL10, IL-23) ( Supplementary Figures S1A–F ), compared to WT groups. Similarly, KO-BMDMs showed a significantly greater inflammatory response to palmitate stimulation, as evidenced by higher mRNA levels of these pro-inflammatory genes, compared to control WT-BMDMs ( Supplementary Figures 2A–I ). In contrast, when stimulated with IL-4, an inducer of M2-like anti-inflammatory response, loss of Lcn10 impaired expression of anti-inflammatory specific genes, including Arg1 ( Figure 2I ), Chil3 ( Figure 2J ), Clec10a ( Figure 2K ), at 6 h or 12 h time point, whereas Retnla mRNA expression from KO BMDMs was increased at 6 h time point ( Figure 2L ). Furthermore, we performed flow cytometry analysis and quantified the percentage of pro-inflammatory (M1-like, CD38^hiCD206^low) ( Figure 2M ) and anti-inflammatory (M2-like, CD38^lowCD206^hi) ( Figure 2M ) macrophages in response to LPS + IFNγ. In line with the gene profiling, we found that Lcn10 deletion shifted BMDMs towards a pro-inflammatory phenotype, as revealed by higher expression levels of CD38 (pro-inflammatory marker) ( Figures 2M, N ), as well as M1/M2 ratio compared with WT-BMDMs ( Figure 2P ); although no difference was observed in CD206 expression (anti-inflammatory marker) between the two groups ( Figures 2M, O ). Taken together, these results suggest that ablation of Lcn10 promotes macrophage pro-inflammatory response while inhibiting the anti-inflammatory response upon multiple stress conditions.

It is well established that macrophage polarization plays an essential role in metabolic disorders and concomitant cardiac dysfunction (44). We were curious to explore whether Lcn10 is involved in diabetes-induced metabolic stress. To this end, we fed WT and Lcn10-KO mice with an HFD for 12 weeks plus one dose of STZ injection at week 4 post-HFD. Mice fed with a chow diet served as controls ( Figure 3A ). Compared with chow-fed mice, HFD feeding for 4 weeks resulted in a significant increase in body weight gain in both WT and Lcn10-KO mice ( Figure 3B ). Consistent with previous reports (45, 46), STZ injection resulted in a moderate weight loss. Accordingly, blood glucose levels in the T2D group increased dramatically after STZ administration when compared to non-diabetic (ND) mice ( Figure 3C ). Interestingly, compared with WT mice, systemic Lcn10 deletion had no significant effect on body weight gain, fed blood glucose, and glucose tolerance in ND and T2D status ( Figures 3B–D ). However, KO-T2D mice clearly displayed impaired insulin sensitivity, as revealed by higher blood glucose levels during the insulin tolerance test compared with WT-T2D controls ( Figure 3E ). Importantly, this effect is independent of body weight gain, indicating that metabolic stress induced by HFD feeding and STZ injection was required for Lcn10 to influence metabolic hemostasis. Lastly, we assessed cardiac function at 8 weeks post-STZ injection using echocardiography. We found that Lcn10-KO mice exhibited normal cardiac function compared with WT controls under chow diet feeding ( Figures 3F, G ). However, compared to WT-T2D mice, KO-T2D mice showed a reduced cardiac contractile function, as evidenced by a 20% reduction in fractional shortening ( Figures 3F, G ). Collectively, these data demonstrate that Lcn10 deficiency leads to aggravated insulin resistance and cardiac dysfunction under diabetic conditions.

Given that the imbalance of macrophage M1/M2 status may contribute to cardiac injury, we next aimed to determine the phenotypes of cardiac macrophages in T2D mice. At the early stage of diabetes (1 week after STZ administration), the flow cytometry results revealed that macrophages in KO-T2D hearts exhibited a more robust pro-inflammatory signature, as evidenced by a higher ratio of Ly6C+ population to CD206+ population compared to WT-T2Ds ( Figures 4A–D ). Such increased inflammatory phenotype in KO-T2D macrophages persisted into the late stage of diabetes, similar results of higher Ly6C+ population and lower CD206+ population were observed at 8 weeks after STZ injection ( Supplementary Figures S3B–E ), which may contribute to the development and progression of cardiac dysfunction under T2D conditions ( Figures 3F, G ). To further confirm the polarized state of cardiac macrophages, we isolated these cells from KO-T2D mice and WT-T2D control mice via magnetic cell sorting, and total RNA was extracted to conduct qPCR for pro- and anti-inflammatory genes. Consistent with our flow cytometry results, macrophages from KO-T2D hearts displayed increased IL-6 ( Figure 4E ), IL-1β ( Figure 4F ), and CCL-2 ( Figure 4H ) expression than those from WT-T2D controls. Interestingly, the expression of anti-inflammatory genes exerted a discrepancy pattern. The mRNA levels of Arg1 ( Figure 4I ) in macrophages from KO-T2D hearts were decreased when compared to WT- T2D groups, while the levels of other anti-inflammatory genes, including Mrc1, Clec10a, and Retnla, were comparable in macrophages from WT- T2D and KO-T2D hearts ( Figures 4J–L ). Altogether, these data suggest that ablation of Lcn10 aggerates macrophage polarization to M1-like phenotype in T2D hearts.

Global ablation of Lcn10 might affect the functions of other vital organs such as the pancreas, liver, or kidney, which may contribute to aggravated diabetic complications. Thus, to further clarify whether Lcn10 deficiency-elicited effects in the heart are mediated by hematopoietic cells or other cells, we performed bone marrow cell transplantation experiments. Specifically, recipient mice received whole-body irradiation to eliminate endogenous hematopoietic cell precursors and were transplanted with bone marrow cells from intact WT and Lcn10-KO mice. Then, these mice were subjected to T2D induction by the combination of HFD and STZ injection ( Figure 5A ). In line with our above results that Lcn10-KO mice displayed exacerbated diabetes-induced cardiac dysfunction, transplantation of Lcn10-deficient bone marrow cells aggravated cardiac dysfunction of recipient mice, evidenced by a 12% decrease in fractional shortening ( Figure 5B ). In addition, transplantation did not affect the percentage of neutrophils (CD45.2+CD11b+Ly6G+) in both recipient groups ( Supplementary Figures S4C, D ). However, repopulating recipient mice with Lcn10-KO cells resulted in an increased number of Ly6C+CD206- pro-inflammatory macrophages in the heart ( Figures 5C–F ). Taken together, these results suggest that Lcn10-KO hematopoietic cells are sufficient to exacerbate diabetes-induced cardiac dysfunction, mainly by triggering more infiltration of pro-inflammatory macrophages in the diabetic heart.

To gain further insights into how the loss of Lcn10 in macrophages induces M1- like phenotype, we isolated BMDMs from WT and Lcn10-KO mice and performed RNA-sequencing analyses. Importantly, we identified that 569 genes were significantly upregulated, whereas 608 genes were remarkably downregulated in KO macrophages compared to WT counterparts ( Figure 6A ). Of interest, further analysis revealed that many of the significantly differentially expressed genes (DEGs) are directly or indirectly regulated by the Nr4a1 signaling pathway ( Figures 6B, C ). More intriguingly, we observed that the gene expression of Nr4a1 itself was significantly decreased in KO macrophages. Notably, it is well appreciated that Nr4a1 acts as a transcriptional activator or repressor depending on post-translational modifications and coregulator protein recruitment (47, 48). In accordance with the RNA-seq data, qRT-PCR analysis further validated the altered expression of Nr4a1-related genes, such as Gdf3, Mid1, Id3, Tgfbi, and Rab4a ( Figure 6D ). Collectively, these data suggest that loss of Lcn10 could disrupt the Nr4a1 signal in macrophages.

Given that Nr4a1 may contribute to the Lcn10-elicited polarization in macrophages, we therefore pre-treated Lcn10-KO BMDMs with CsnB, a specific agonist of Nr4a1, 30 minutes before LPS+IFNγ stimulation. Subsequently, the gene expression of pro-inflammatory markers was analyzed. As expected, we observed that treatment of Lcn10-KO macrophages with CsnB significantly inhibited pro-inflammatory response upon LPS+IFNγ stimulation, compared to controls, as measured by mRNA levels of iNOS, IL-6, IL-1β, and CXCL10 ( Figures 7A–D ). Previous studies have revealed that the Nr4a1-modulated expression of its target genes is primarily ascribed to its transcriptional activity (19, 22). Hence, to dissect the potential mechanism underlying the effect of Lcn10 deficiency in Nr4a1 signaling, we determined the translocation of Nr4a1 in macrophages from WT and KO mice using immunofluorescence staining. Under basal status, Nr4a1 was distributed in cytosol and nucleus, which is similar in WT and KO macrophages ( Figure 7E ). However, when treated with LPS+IFNγ, WT-BMDMs showed a substantial increase in the nuclear translocation of Nr4a1 when compared with controls ( Figure 7E ). Notably, such nuclear translocation was attenuated in KO-BMDMs as more Nr4a1 was retained in the cytosol ( Figure 7E ). Consistently, qRT-PCR analysis validated that such distribution change affected the expression of several Nr4a1-targeted genes in KO-BMDMs treated with LPS+IFNγ: with down-regulation of anti-inflammatory gene (Gdf3, Tgfbi) ( Figure 7F ), whereas up-regulating pro-inflammatory gene Rab4a ( Figure 7F ). Accordingly, CsnB treatment partially improved cardiac function in Lcn10-KO mice under T2D conditions, yet to a lesser extent when comparing to WT groups ( Figures 7G, H ). Taken together, these data indicate that disruption of the Nr4a1 signal may contribute to Lcn10 KO-mediated inflammatory responses in macrophages and subsequent cardiac injury.