Complement 3a Receptor 1 on Macrophages and Kupffer cells is not required for the Pathogenesis of Metabolic Dysfunction-Associated Steatotic Liver Disease

Together with obesity and type 2 diabetes, metabolic dysfunction-associated steatotic liver disease (MASLD) is a growing global epidemic. Activation of the complement system and infiltration of macrophages has been linked to progression of metabolic liver disease. The role of complement receptors in macrophage activation and recruitment in MASLD remains poorly understood. In human and mouse, C3AR1 in the iver is expressed primarily in Kupffer cells, but is downregulated in humans with MASLD compared to obese controls. To test the role of complement 3a receptor (C3aR1) on macrophages and liver resident macrophages in MASLD, we generated mice deficient in C3aR1 on all macrophages (C3aR1-MφKO) or specifically in liver Kupffer cells (C3aR1-KpKO) and subjected them to a model of metabolic steatotic liver disease. We show that macrophages account for the vast majority of C3ar1 expression in the liver. Overall, C3aR1-MφKO and C3aR1-KpKO mice have similar body weight gain without significant alterations in glucose homeostasis, hepatic steatosis and fibrosis, compared to controls on a MASLD-inducing diet. This study demonstrates that C3aR1 deletion in macrophages or Kupffer cells, the predominant liver cell type expressing C3aR1, has no significant effect on liver steatosis, inflammation or fibrosis in a dietary MASLD model.


Introduction
Obesity and related metabolic diseases such as type 2 diabetes (T2D) and metabolic dysfunction-associated steatotic liver disease (MASLD) remain a worldwide epidemic with increasing prevalence 1,2 .MASLD describes the constellation of hepatic lipid deposition, inflammation, and fibrosis associated with obesity and T2D that ultimately leads to MASH cirrhosis, which has become the leading cause of liver transplantation in the United States 3-6 .
Notably, MASLD is increasingly recognized as an important risk-enhancing factor for atherosclerotic cardiovascular disease 7,8 .
Liver macrophages help to maintain hepatic homeostasis and consist of embryo-derived resident macrophages called Kupffer cells, which self-renew and do not migrate, or peripheral monocyte-derived macrophages, which infiltrate into liver tissue upon metabolic or toxic liver injury and under certain circumstances can take on Kupffer cell-like identity [9][10][11][12][13] .In obesity, bone marrow-derived myeloid cells migrate to the steatotic liver, and pro-inflammatory recruited macrophages are postulated to drive the progression of MASLD to MASH 14 .Spatial proteogenomics reveals a population of lipid-associated macrophages near bile canaliculi that is induced by local lipid exposure and drives fibrosis in steatotic regions of murine and human liver 15 .In addition, deep transcriptomic profiling in human MASLD has identified candidate gene signatures for steatohepatitis and fibrosis with possible therapeutic implications 16 .
Activation of the body's complement system leads to increased cell lysis, phagocytosis, and inflammation 17 , and it is increasingly recognized as an important contributor to regulation of metabolic disorders such as T2D and MASLD 18,19 .In human liver biopsies, higher lobular inflammation scores correlate with activation of the complement alternative pathway 20 , which can signal via the C3a receptor 1 (C3aR1), a G i -coupled G protein-coupled receptor 21 .The complement 3 polypeptide (C3) is cleaved by C3 convertase to the activated fragment, C3a, which then binds C3aR1 22 .Complement factor D (CFD), also known as the adipokine adipsin, is the rate-limiting step in the alternative pathway of complement activation 23,24 .
Several studies have reported opposing roles of adipsin and C3aR1 on hepatic steatosis in diet-induced obesity [25][26][27] .Our lab has found that adipsin/CFD is critical for maintaining pancreatic beta cell mass and function 28,29 .Murine obese and diabetic models such as db/db mice and high fat diet (HFD) feeding result in very low circulating adipsin 23 .Replenishing adipsin in db/db mice raises levels of C3a and insulin, lowers blood glucose levels, and inhibits hepatic gluconeogenesis 28 .However, whole-body deletion of C3aR1 decreases macrophage infiltration and activation in adipose tissue, protects from HFD-induced obesity and glucose intolerance, and decreases hepatic steatosis and inflammation 30 .In a model of fibrosing steatohepatitis, bone marrow-derived macrophages were found to activate hepatic stellate cells, which was blunted in whole-body C3aR1 KO mice 31 .
In the present study we aim to explore the macrophage-specific effect of complement receptor signaling in MASLD pathogenesis.To determine the consequences of macrophage and Kupffer cell ablation of C3aR1, we use a murine dietary model of MALFD/MASH, the Gubra Amylin Nash (GAN) diet, which has macronutrient similarities to the Western diet and produces similar histologic and transcriptomic changes to human MASLD/MASH [32][33][34] .

C3AR1 is expressed in human and mouse liver, primarily in Kupffer cells.
In the scRNA-Seq database, Human Protein Atlas, C3AR1 is broadly expressed throughout the body, with increased abundance in tissues rich in immunologic cell types, such as bone marrow and appendix (Fig. 1A) 35 .In a single-cell transcriptomic database of healthy human liver, C3AR1 expression predominates in the macrophage and Kupffer cell population, with minimal-to-undetectable C3AR1 expression in hepatocytes or hepatic stellate cells by scRNA-Seq (Fig. 1B) 36 .In the mouse liver scRNA-Seq database, Tabula Muris, C3ar1 is similarly expressed primarily in Kupffer cells (Fig. S1) 37 . .

Hepatic CFD and C3AR1 are downregulated in human MASLD/MASH.
We also examined data from Suppli and coworkers, who performed bulk transcriptomic analysis of human liver samples from an age-matched cohort of healthy controls and obese controls without MASLD, as well as MASLD and MASH patients without cirrhosis 38 .Both CFD and C3AR1 were unchanged in obese subjects without MASLD compared to healthy controls, but both CFD and C3AR1 were significantly downregulated in liver biopsies from both MASLD and MASH patients compared to both healthy controls and obese subjects without MASLD (Fig. 1C).Interestingly, both CFD and C3AR1 levels were slightly higher in MASH individuals compared to those with MASLD only.

Murine MASH model recapitulates key features of human MASH
At 5 weeks of age, we subjected C3ar1 flox/flox control mice to standard regular diet (RD) or GAN diet l 32,33 .After 28 weeks of GAN diet, male mice gained body weight compared to RD (Fig. 1D), primarily as fat mass (Fig. S2-3), but weight gain in female GAN-fed mice was attenuated.Histologic signs of MASLD were present in GAN-fed mice (Fig. 1E), most notably hepatic steatosis and hepatocyte ballooning (Fig. 1F), and liver fibrosis measured by collagen deposition nearly doubled with GAN compared to RD (Fig. 1G).Both hepatic C3ar1 and Cfd gene expression were robustly increased on GAN compared to RD, as were markers of macrophage infiltration, hepatic inflammation, and fibrosis, including collagen gene expression, indicating progression to fibrotic MASH (Fig. 1H).

Macrophage-specific C3aR1 deletion does not alter glucose homeostasis.
Owing to the differential regulation of the C3AR1 gene in MASLD between mice and humans, we generated transgenic mice with macrophage-specific deletion of C3aR1 (C3aR1-MKO) to target both liver resident macrophages and recruited monocytes.Successful deletion of C3ar1 in macrophages from the C3aR1-MKO mouse was confirmed by quantitative RT-PCR of isolated peritoneal macrophages that were F4/80+ and CD68+ by fluorescenceactivated cell sorting (Fig. 2A).In liver tissue, C3ar1 expression was reduced by ~88% in both male and female C3aR1-MKO (Fig. 2B).These results indicate that macrophages account for the vast majority of C3ar1 expression in the liver.
When placed on GAN diet, there was no significant difference in weight gain between control and C3aR1-MKO mice (Fig. 2C).There was similarly no difference in percent lean or fat mass between these mice (Fig. 2D).Glucose tolerance tests performed in fasted mice after 27 weeks GAN diet found no significant differences between control and C3aR1-MKO mice (Fig. 2E).There was also no difference in insulin sensitivity as measured by insulin tolerance tests in male mice (Fig. S4).Insulin resistance as measured by comparing the ratio of fasting glucose level to fasting insulin level (HOMA-IR) was also unchanged between controls and C3aR1-MKO mice (Fig. S5).Circulating serum ALT levels were unchanged in male control and C3aR1-MKO mice on GAN diet (Fig. S6).

Macrophage-specific C3aR1 deletion does not significantly impact hepatic steatosis or fibrosis.
Liver samples collected after 28-30 weeks of GAN or regular diet did not show significant differences in liver mass between control and C3aR1-MKO mice (Fig. 2F).Male mice on GAN diet developed similar qualitative appearance on histology (Fig. 2G), and slide image analysis showed similar proportions of lipid droplet area and collagen area (Figs.2H, 2I).This indicates that there were no significant differences in steatosis or fibrosis between GAN-fed control and C3aR1-MKO male mice.While C3ar1 expression was markedly reduced in the C3aR1-MKO liver tissue (Fig. 2B), there were no detectable gene expression changes in markers of fibrosis, inflammation, or lipid handling (Fig. 2J).Similarly, in female mice there were also no significant differences between control and C3aR1-MKO mouse liver in a subset of key gene markers of fibrosis or inflammation (Fig. S7).

Kupffer cell-specific C3aR1 deletion does not alter weight gain or glucose homeostasis.
To explore whether there may be competing effects between recruited monocytes and liver resident macrophages (Kupffer cells), we next generated Kupffer cell-specific C3aR1 knockout mice (C3aR1-KpKO) and fed them GAN diet.Body weight gain was similar between genotypes for both male and female mice (Fig. 3A), and there was no difference in body composition between control and C3aR1-KpKO mice on GAN diet (Fig. 3B).There was similarly no significant difference in glucose homeostasis between the genotypes during a glucose tolerance test (Fig. 3C).

Kupffer cell-specific C3aR1 deletion does not significantly impact hepatic steatosis or fibrosis.
Liver mass was not significantly different between control and C3aR1-KpKO mice on GAN diet (Fig. 3D).Liver sections appeared qualitatively similar by histology stained with Masson's trichrome (Fig. 3E).There were similar levels of hepatic steatosis in these mice as measured by percent lipid droplet area (Fig. 3F).When measured by collagen proportional area, there was no significant differences in liver fibrosis between C3aR1-KpKO and control mice (Fig. 3G).While C3ar1 expression was reduced by 73% in liver tissue of C3aR1-KpKO mice, there were no significant differences in expression of inflammatory, fibrotic, or lipid handling gene markers (Fig. 3H).C3ar1 expression similarly decreased by ~90% in liver tissue of female C3aR1-KpKO mice fed regular diet compared to control mice (Fig. S8).These data also indicate that Kupffer cells account for ~80% of hepatic C3ar1 gene expression in our mouse model of MASLD/MASH.

Discussion
Overall, we found that macrophage or Kupffer cell expression of C3ar1 does not impact body weight gain or histologic/transcriptomic features of MASLD/MASH in a murine dietary .model.Deletion of C3aR1 in the macrophage population throughout the body, or specifically in Kupffer cells, did not affect weight gain, glucose homeostasis, or extent of hepatic steatosis/fibrosis.
Our findings in macrophage-specific C3aR1 KO mice contrast with prior observations in whole-body C3aR1 KO mice 30 , which are protected from diet-induced obesity, have improved glucose tolerance, and exhibit decreased hepatic steatosis.In both our macrophage-and Kupffer cell-specific C3aR1 KO mice, which had similar degrees of obesity compared to controls, there was no detectable effect on liver steatosis or fibrosis despite the near abrogation of C3ar1 expression.This raises the possibility that the lower levels of hepatic steatosis and insulin resistance previously observed in the whole body C3aR1 KO mice may be secondary to protection from obesity.Protection from diet-induced obesity in whole-body C3aR1 KO mice may be mediated by a non-macrophage cell type, since our macrophage-specific C3aR1 KO mice were not afforded this protection.The C3ar1-expressing cell types that promote obesity and MASLD remains to be determined.
Our laboratory recently reported sex-dependent regulation of thermogenic adipose tissue mediated by adipocyte-derived C3aR1 39 .However, no such sexual dimorphism was observed in hepatic expression of key MASH genes in response to GAN diet in our macrophage-or Kupffer cell-specific C3aR1-deficient mice.Other work has suggested possible compensatory effects from its sister anaphylatoxin receptor C5aR1, with increased cold-induced adipocyte browning and attenuated diet-induced obesity seen in C3aR1/C5aR1 double KO mice 40 .
The strengths of our study include careful metabolic and transcriptomic phenotyping of cell type-specific transgenic mice.Some limitations were our use of a single MASLD dietary model and our focus on the C3aR1 pathway.While the GAN diet recapitulates many features of human MASH due to its similarity to Western diet 34 , relatively low levels of fibrosis were seen in our study, potentially related to initiating the diet at young age; more rapid fibrosis induction has been seen when GAN diet is initiated at older ages 41

Animals
C3ar1 flox/flox mice were on the C57BL/6J background as described 42 .Homozygous LysM-Cre mice on the C57BL/6J background were purchased from Jackson Laboratories (Strain #004781).C3ar1 flox/flox homozygous mice were used in the experiments as controls from the same backcross generation 39 .All mice were maintained in plastic cages under a 12h/12h light/dark cycle at constant temperature (22°C) with free access to water and food.

Blood chemistry and serum insulin analysis
. Mice were fasted overnight (14-16 hours) for glucose tolerance tests and injected intraperitoneally with syringe-filtered D-glucose solution (2g/kg).For insulin tolerance test, mice were fasted for 6 hours and injected with 0.5 mIU/kg insulin.Blood glucose levels were assayed by commercial glucometer (OneTouch) by tail vein blood samples.Plasma insulin levels were measured from mice fasted for 6 hours.Tail vein blood was collected into lithium heparin-coated tubes, centrifuged at 2000xg at 4ºC, and plasma insulin levels were determined by ELISA using a standard curve (Mercodia).Serum alanine aminotransferase levels were measured in serum from blood collected via cardiac puncture using a commercially available colorimetric assay (TR71121, ThermoFisher Scientific).

Peritoneal macrophage isolation and flow cytometry
Peritoneal macrophages were isolated from as previously described 43 .Briefly, mice were euthanized then immediately injected intraperitoneally with 10 mL phosphate-buffered saline (PBS, pH 7.4) at room temperature.After a 3-5 minute incubation period, peritoneal fluid was removed with sterile needle and syringe and placed on ice.After centrifugation at 300xg, the pellet was resuspended in PBS containing 2% fetal bovine serum and 0.1% sodium azide.

Histological studies
A mid-distal portion of the left liver lobe was fixed with 10% buffered formalin and transferred to 70% ethanol.Samples were embedded in paraffin, sectioned at ~5m thickness, and stained with Masson's trichrome.Slides were imaged using Zeiss Axioscan7 at 20x magnification.Histologic analyses were performed using ImageJ software (version 1.53t).Lipid .

S1)
Single cell RNA sequencing analysis of C3ar1 expression in mouse liver tissue (see text).

S2)
Percent lean and fat mass of flox/flox control mice after 20 weeks of GAN or RD diet (n = 6-7 per group).

S3)
Absolute lean and fat mass of flox/flox control mice after 20 weeks of GAN or RD diet (n = 6-7 per group).

S4)
Insulin tolerance test in control or C3aR1-MKO male mice with 14h fast after 29 weeks GAN diet (n = 6-9 per group).

S5)
HOMA-IR measurement of insulin resistance in control or C3aR1-MKO mice with 6h fast after 27 weeks GAN diet (n = 6-9 per male group, n = 9-13 per female group).

S6)
Serum alanine aminotransferase levels in control or C3aR1-MKO male mice after 30 weeks GAN diet (n = 4 per group).

S7)
Relative gene expression in control or C3aR1-MKO female mice after 30 weeks GAN diet (n = 13-14 per group).

Supplementary Table
. Lastly, while C3AR1/C3ar1 expression is very low in non-macrophage cells (Fig. B, S1), C3aR1 signaling on other hepatic cell types not explored in this study, such as hepatic stellate cells, could mediate the observed effect in the whole-body C3aR1 KO mouse.Deletion of C3aR1 in macrophages generally, or in liver resident macrophages specifically, had no major effect on systemic glucose homeostasis and hepatic steatosis, inflammation, and fibrosis in this murine dietary model of MASLD/MASH.The complement system is a complex entity directing an important part of the body's inflammatory and tissue repair response in MASLD.Further work is needed to elucidate the mechanisms of the role of C3aR1 in the pathogenesis of MASH and cirrhosis.

Figure 1 .
Figure 1.C3AR1 is found in macrophages, is modulated by MASLD/MASH in humans, and is induced by a murine dietary model of MASH.

Figure 2 .
Figure 2. C3aR1 deletion in all macrophages does not affect weight gain, glucose homeostasis, liver steatosis or fibrosis.

Figure 3 .
Figure 3. C3aR1 deletion in Kupffer cells does not affect weight gain, glucose homeostasis, liver steatosis or fibrosis.