GPR183 antagonism reduces macrophage infiltration in influenza and SARS-CoV-2 infection

Cheng Xiang Foo, Stacey Bartlett, Keng Yih Chew, Minh Dao Ngo, Helle Bielefeldt-Ohmann, Buddhika Jayakody Arachchige, Benjamin Matthews, Sarah Reed, Ran Wang, Christian Smith, Matthew J. Sweet, Lucy Burr, Kavita Bisht, Svetlana Shatunova, Jane E. Sinclair, Rhys Parry, Yuanhao Yang, Jean-Pierre Lévesque, Alexander Khromykh, Mette Marie Rosenkilde, Kirsty R. Short, Katharina Ronacher
European Respiratory Journal 2022; DOI: 10.1183/13993003.01306-2022

Abstract

Rationale Severe viral respiratory infections are often characterised by extensive myeloid cell infiltration and activation and persistent lung tissue injury. However, the immunological mechanisms driving excessive inflammation in the lung remain poorly understood.

Objectives To identify the mechanisms that drive immune cell recruitment in the lung during viral respiratory infections and identify novel drug targets to reduce inflammation and disease severity.

Methods Preclinical murine models of influenza virus and severe acute respiratory coronavirus 2 (SARS-CoV-2) infection.

Results Oxidised cholesterols and the oxysterol-sensing receptor GPR183 were identified as drivers of monocyte-macrophage infiltration to the lung during influenza virus (IAV) and SARS-CoV-2 infection. Both IAV and SARS-CoV-2 infection upregulated the enzymes cholesterol 25-hydroxylase (CH25H) and cytochrome P450 family 7 subfamily member B1 (CYP7B1) in the lung, resulting in local production of the oxidised cholesterols 25-hydroxycholesterol (25-OHC) and 7α,25-dihydroxycholesterol (7α,25-OHC). Loss-of-function mutation of GPR183, or treatment with a GPR183 antagonist, reduced macrophage infiltration and inflammatory cytokine production in the lungs of IAV- or SARS-CoV-2-infected mice. The GPR183 antagonist significantly attenuated the severity of SARS-CoV-2 infection and viral loads. Analysis of single cell RNASeq data on bronchoalveolar lavage samples from healthy controls and COVID-19 patients with moderate and severe disease revealed that CH25H, CYP7B1 and GPR183 are significantly upregulated in macrophages during COVID-19.

Conclusion This study demonstrates that oxysterols drive inflammation in the lung via GPR183 and provides the first preclinical evidence for therapeutic benefit of targeting GPR183 during severe viral respiratory infections.

Introduction

Severe viral respiratory infections including influenza and COVID-19 are associated with extensive myeloid cell recruitment to the lung, which can lead to severe tissue injury and the development of acute respiratory distress syndrome (ARDS) [1]. A shift in lung macrophage composition and function is associated with COVID-19 severity. A study of >600 hospitalised patients found that in severe cases resident alveolar macrophages were depleted and replaced by large numbers of inflammatory monocyte-derived macrophages [2]. Rapid monocyte-macrophage infiltration of the lung during the acute phase of severe acute respiratory coronavirus 2 (SARS-CoV-2) infection is replicated in animal models [3, 4].

Oxidised cholesterols have recently emerged as markers of inflammation in the lung. Oxysterols were increased in bronchoalveolar lavage fluid (BALF) from inflamed airways after allergen challenge and correlated with infiltrating leukocytes [5]. They were also increased in the sputum from patients with chronic obstructive pulmonary disease correlating with disease severity [6, 7] and in the lungs of mice after lipopolysaccharide (LPS)-induced lung inflammation [8]. However, the role of oxysterols in the lung during viral respiratory infections has not been investigated.

Oxysterols have a range of receptors sharing a common role in inflammation [9]. One oxysterol pathway leads to the production of 7α,25-hydroxycholesterol (7α,25-OHC), via cholesterol 25-hydroxylase (CH25H) and cytochrome P450 family 7 subfamily B member 1 (CYP7B1) [9, 10]. 7α,25-OHC can subsequently be metabolised by hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta isomerase7 (HSD3B7) (figure 1a). 7α,25-OHC is the endogenous high affinity agonist of the oxysterol-sensing G protein-coupled receptor GPR183 [11, 12]. GPR183 is expressed on cells of the innate and adaptive immune systems, including macrophages, dendritic cells, innate lymphoid cells, eosinophils and T and B lymphocytes [5, 13, 14]. With its oxysterol ligands GPR183 facilitates the chemotactic distribution of immune cells to secondary lymphoid organs [9, 11, 13, 15]. In vitro GPR183 mediates migration of human and mouse macrophages towards a 7α,25-OHC gradient [16, 17].

FIGURE 1
FIGURE 1

IAV infection leads to upregulation of CH25H and CYP7B1 expression in the lung and production of the oxysterols 25-OHC and 7α,25-OHC. a) The biosynthetic pathway of 25-OHC and 7α,25-OHC. b) Experimental design; C57BL/6J mice were infected intranasally with 5500 PFU of IAV and mRNA expression of (c) Ch25h, Cyp7b1 and Hsd3b7 were measured by qRT-PCR at 3 dpi and 7 dpi normalised to Hprt (upper panel). Quantitative analysis of CH25H, CYP7B1 and HSD3B7 protein labelling by IHC (lower panel). Representative IHC images of CH25H, CYP7B1 and HSD3B7 in lung sections of uninfected or IAV-infected mice. d) Concentrations of 25-OHC and 7α,25-OHC in the lungs at 3 dpi and 7 dpi expressed in pmol per gram lung tissue (left panel) or in pmol per mL BALF (right). Data are presented as mean } sd of n=4 uninfected and n=6–10 infected mice per timepoint.

In this study, we hypothesised that viral respiratory infections lead to the production of oxysterols in the lung contributing to excessive immune cell infiltration and inflammation. We show that oxysterols drive GPR183-dependent monocyte-macrophage infiltration in preclinical models of IAV and SARS-CoV-2 infection and identify GPR183 is a host target for therapeutic intervention to mitigate disease severity in viral respiratory infections.

Methodology

Ethics and biosafety

All experiments were approved by the Animal Ethics Committee (MRI-UQ/596/18, AE000186) and the Institutional Biosafety Committee of the University of Queensland (IBC/465B/MRI/TRI/AIBN/2021).

Viral strains

Virus stocks of A/H1N1/Auckland/1/2009(H1N1), referred to as influenza A virus (IAV), were prepared in embryonated chicken eggs. A mouse-adapted SARS-CoV-2 strain was obtained through serial passage of SARS-CoV-2 (B.1.351; hCoV-19/Australia/QLD1520/2020, GISAID accession EPI_ISL_968081, collected on 29 December 2020, kindly provided by Queensland Health Forensic and Scientific Services). A description of the mouse-adaption and genomic sequencing data for the SARS-CoV-2 strain can be found in the supplemental methods (Figure S1). IAV viral titres were determined by plaque assays on Madin-Darby canine kidney (MDCK) and SARS-CoV-2 plaque assays on Vero E6 cells as described in the supplement.

Mouse models

Gpr183tm1Lex were obtained from Lexicon Pharmaceuticals (The Woodlands, USA), back-crossed to a C57BL/6J background and bred in-house. Eight to 10-week-old C57BL/6J and Gpr183tm1Lex (Gpr183−/−) mice were anesthetised with 4% isoflurane and infected intranasally with 5,500 PFU of IAV A/Auckland/01/09 (H1N1). For SARS-CoV-2 infection, C57BL/6J and Gpr183−/− mice were anesthetised with ketamine/Xylazine (80 mg·kg−1/5 mg·kg−1) and infected intranasally with 8×104 PFU of mouse-adapted SARS-CoV-2. Lungs were collected at specified timepoints for subsequent downstream analysis as described in supplemental data. The GPR183 antagonist NIBR189 (7.6 mg·kg−1) in vehicle (0.5% carboxymethylcellulose/0.5% Tween-80) or vehicle only was administrated by oral gavage from 1 dpi, twice daily at 12-hour intervals until the end of the experiment.

RNA isolation and RT-qPCR

Total RNA was isolated using ISOLATE II RNA Mini Kit (Bioline Reagents Ltd., London, UK) as previously described [18, 19]. The list of primers is provided in Table S1. The relative expression (RE) of each gene normalised to the reference gene (Hypoxanthine-guanine phosphoribosyltransferase; Hprt) was determined using the 2−ΔCt method.

Oxysterol extraction and mass spectrometric quantitation

The oxysterol extraction and quantification methods were adapted from Ngo et al. [18] as described in the supplemental information.

Cytokine quantification using ELISA

Cytokines in lung homogenates were measured with DuoSet ELISA (IFNβ (DY8234-05), IFNγ (DY485), IFNλ (DY1789B), IL-6 (DY406), TNFα (DY410), IL-1β (DY401), IL-10 (DY417) and CCL2 (DY479), R&D systems) according to the manufacturer's protocol.

Flow cytometry

Flow cytometry was performed on single cell suspensions from digested lungs and blood as described in detail in the supplement.

Immunohistochemistry

Immunohistochemistry (IHC) was performed on deparaffinised/rehydrated lung sections by immunolabelling with antibodies against SARS-CoV-2 nucleocapsid protein (40143-R040 Sino Biological), IBA1 (019-19741; NovaChem), CH25H (BS-6480R, Bioss Antibodies), CYP7B1 (BS-5052R, Bioss Antibodies) and isotype control (rabbit IgG 31235, Thermo Fisher Scientific) diluted in Da Vinci Green Diluent (PD900, Biocare Medical) followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit Ig antibody (1:200) (ab6721, Abcam). See supplementary data for details. Isotype controls are shown in (Figure S2).

Statistical analysis

Data were analysed on GraphPad Prism software. Data were assessed for normality using Shapiro-Wilk test. Spearman rank correlation was used to analyse correlations. For two group comparisons, parametric Student's two-tailed t-test was used for normally distributed data while nonparametric Mann-Whitney U test was used for data that deviate from normality.

Results

IAV infection increases CH25H and CYP7B1 expression and oxysterol production in the lung

To investigate whether IAV infection induces the production of oxidised cholesterols, mice were infected with IAV (figure 1b) and mRNA expression of oxysterol-producing enzymes was determined in lung tissue. Ch25h and Cyp7b1 mRNA were increased in lungs of IAV-infected mice compared to uninfected animals, whereas Hsd3b7 was downregulated in the lung 7 days post infection (dpi) (figure 1c, upper panel). Similarly, CH25H and CYP7B1 proteins were also increased while HSD3B7 remained constant, as demonstrated by immunohistochemical labelling of lung sections with antibodies detecting CH25H, CYP7B1 and HSD3B7 (figure 1c, lower panel). The induction of oxysterol-producing enzymes was associated with increased concentrations of the oxysterols 7α,25-OHC and 25-OHC in lung homogenates (figure 1d, left panels) and bronchoalveolar lavage fluid (BALF) (figure 1d, right panels) from IAV-infected animals at both 3 and 7 dpi. In uninfected lungs, 7α,25-OHC was undetectable in most samples tested. Ch25h and Cyp7b1 mRNA were increased in the bronchoalveolar lavage (BAL) cell pellet of IAV-infected mice compared to uninfected animals while Hsd3b7 remained unchanged (Figure S3a).

Consistent with the increase in oxysterols, Gpr183 mRNA was increased at 3 and 7 dpi in both BAL cells (Figure S3a) and lung tissue (Figure S3b), suggesting increased expression and/or recruitment of GPR183-expressing immune cells to the lung upon infection. Gpr183 expression was positively correlated with Ch25h and Cyp7b1 (Figure S3c).

Gpr183−/− mice have reduced macrophage infiltration into the lungs upon IAV infection

To investigate whether oxysterol-mediated immune cell recruitment is dependent on the oxysterol-sensing GPR183, we performed experiments in mice genetically deficient in Gpr183 (Gpr183−/−). Gpr183−/− mice exhibit normal gross phenotype [20], had normal circulating monocyte numbers and comparable numbers of macrophage colony forming units (CFU-M) in the bone marrow to C57BL/6 mice (Figure S4) suggesting comparable monopoiesis in Gpr183−/− mice. However, upon infection with IAV, Gpr183−/− mice had lower IBA1+ macrophage numbers in the lung at 3 and 7 dpi compared to infected C57BL/6J controls (figure 2a). Gpr183 expression was positively correlated with mRNA expression of the pro-inflammatory cytokines Il6, Tnf and Ccl2 in C57BL/6J mice (Figure S5) and reduced macrophage infiltration in Gpr183−/− mice was associated with reduced Il6 and Tnf, but not Ccl2 at 7 dpi (Figure S6). Body weights and viral titres through the course of IAV infection were comparable across the genotypes (Figure S7). These results demonstrate that lack of GPR183 reduces macrophage infiltration into the lung upon IAV infection which is associated with reduced pro-inflammatory cytokine expression.

FIGURE 2
FIGURE 2

Deletion of the Gpr183 gene or administration of a GPR183 antagonist reduces macrophage infiltration in IAV-infected lungs. C57BL/6J and Gpr183-/- mice were infected intranasally with 5500 PFU of IAV. a) Representative IHC images of IBA1 in lung sections of IAV-infected C57BL/6J and Gpr183-/- mice. Quantitative analysis of IBA1 staining. b) Experimental design; C57BL/6J mice and Gpr183−/− mice were infected intranasally with 5500 PFU of IAV. Mice were subsequently treated orally with 7.6 mg·kg−1 NIBR189 or vehicle control twice daily from 1 dpi until the end of the experiment. c) Representative IHC images of IBA1 in lung sections of C57BL/6J and Gpr183-/- mice with the respective treatment groups at 3 dpi and 7 dpi. Quantitative analysis of IBA1 staining. Data are presented as mean } sd of n=6–12 infected mice per genotype and timepoint. dpi=days post-infection; Scale Bar=100 μm; U/I=mock infected ns=not significant; *, p<0.05; **, p<0.01; ***, p<0.001 indicate significant differences.

GPR183 antagonism reduces macrophage infiltration

To investigate whether GPR183 is a putative therapeutic target to reduce inflammation, the GPR183 antagonist NIBR189 [11, 17] was administered to C57BL/6J mice twice daily starting from 24 h post-infection until the end of the experiment (figure 2b). Like Gpr183−/− mice, C57BL/6J animals treated with NIBR189 had significantly reduced macrophage infiltration into the lung both at 3 and 7 dpi as determined by IHC (figure 2c).

In addition, flow cytometry analysis was performed on lung single cell suspensions from C57BL/6J and Gpr183−/− mice treated with NIBR189 and vehicle, respectively, using a previously published gating strategy [21] (Figure S8). NIBR189-treated C57BL/6J mice and Gpr183−/− mice had lower percentages of recruited/infiltrated macrophages (F480high/CD11b+/Ly6G−/SigF−) (figure 3a) compared to vehicle-treated C57BL/6J animals after IAV infection. NIBR189 treatment did not change the percentages of other immune cell subsets in the lung, including neutrophils (B220−/CD3−/Ly6G+/CD11b+) (figure 3a), CD4+ T cells, CD8+ T cells, B cells, DCs, and alveolar macrophages (Figure S9, S10). Body weights and lung viral loads were not affected by genotype or treatment (Figure S11).

FIGURE 3
FIGURE 3

The GPR183 antagonist NIBR189 reduces macrophage infiltration and inflammatory cytokine production. C57BL/6J and Gpr183−/− mice were infected intranasally with 5500 PFU of IAV. Mice were subsequently treated orally with 7.6 mg·kg−1 NIBR189 or vehicle control twice daily from 1 dpi until the end of the experiment. a) Frequency of infiltrating macrophages (F480high/CD11b+/Ly6G−/SigF−) and neutrophils (B220−CD3−Ly6G+) was determined by flow cytometry relative to total viable CD45+ immune cells at 3 dpi. Graphs show the frequency of macrophages and neutrophils. b) Cytokine measurements of IL-6, TNF, IFNβ and IFNλ at 7 dpi measured by ELISA. Data are presented as mean } sd of n=5–12 infected mice per genotype and timepoint. dpi=days post-infection; U/I=mock infected; ns=not significant; *, p<0.05; **, p<0.01 indicate significant differences.

These results demonstrate that NIBR189 significantly reduced macrophage infiltration to the lung without affecting the recruitment of other immune cell subsets.

GPR183 antagonism reduces IAV-induced pro-inflammatory cytokine concentrations

We next determined if the reduced macrophage infiltration mediated by NIBR189 results in reduced inflammatory cytokine production in the lung. At 3 dpi, no significant differences in cytokine production were observed between treatment groups (Figure S12). However, IAV-Infected C57BL/6J mice treated with NIBR189 had significantly lower concentrations of IL-6, TNF and IFNβ (figure 3b) at 7 dpi. This was comparable to the phenotype of IAV-infected Gpr183−/− mice, with NIBR189 treatment having no additional effect in mice deficient in GPR183. In addition, no significant differences were observed in IFNλ across the two timepoints (figure 3b and Figure S12) demonstrating that the GPR183 antagonist treatment does not negatively impact the production of type III IFNs which are important for viral control in the lung [22]. No differences between treatment groups were observed at either timepoint for protein concentrations of IL-1β, CCL2 or IFNγ between treatment groups (Figure S12 and S13). Thus, GPR183 can be inhibited pharmacologically to reduce proinflammatory cytokines upon severe IAV infection.

GPR183 antagonism reduces SARS-CoV-2 infection severity

Excessive macrophage infiltration and activation is a hallmark of severe COVID-19 [2, 23]. To evaluate whether the benefits of inhibiting GPR183 extend to SARS-CoV-2 infection, we established a mouse-adapted SARS-CoV-2 strain by passaging the Beta variant of SARS-CoV-2 (B.1.351) four times in C57BL/6J mice. This resulted in a virus that contained a mutation in NSP5 and caused clinical signs (weight loss) in infected mice (Figure S1). Consistent with the IAV infection results, mRNA expression of Ch25h and Cyp7b1 was significantly upregulated in the lungs of SARS-CoV-2 infected mice compared to uninfected mice while Hsd3b7 remained unchanged (figure 4a). This was confirmed also at the protein level by IHC (figure 4b,c). Further, 25-OHC and 7α,25-OHC concentrations in lung homogenates (figure 4d, left panels) and BALF (figure 4d, right panels) were significantly increased at 2 dpi, returning to uninfected levels by 5 dpi by which time the animals began to recover from the infection. Ch25h and Gpr183 was also increased in BAL cells of SARS-CoV-2-infected mice while Hsd3b7 remained unchanged (Figure S14). NIBR189 or vehicle was administered to C57BL/6J or Gpr183−/− mice twice daily from 24 h post-SARS-CoV-2 infection until the end of the experiment (figure 5a). NIBR189-treated C57BL/6J mice lost significantly less weight and recovered faster compared to vehicle treated mice (figure 5b and Figure S15). Similarly, Gpr183−/− mice had less severe SARS-CoV-2 infection. Collectively, these data demonstrate that oxysterols are produced in the lung upon SARS-CoV-2 infection and GPR183 antagonism significantly reduced SARS-CoV-2 infection severity.

FIGURE 4
FIGURE 4

SARS-CoV-2 infection leads to upregulation of CH25H and CYP7B1 expression in the lung and production of the oxysterols 25-OHC and 7α,25-OHC. C57BL/6J mice were infected intranasally with approximately 8×104 PFU of mouse49 adapted SARS-CoV-2. mRNA expression of (a) Ch25h, Cyp7b1 and Hsd3b7 was measured by qRT-PCR at 2 dpi and 5 dpi normalised to Hprt. b) Quantitative analysis of CH25H, CYP7B1 and HSD3B7 protein by IHC labelling and (c) representative IHC images of CH25H, CYP7B1 and HSD3B7 in lung sections in uninfected mice and 2 and 5 dpi. d) Concentrations of 25-OHC and 7a,25-OHC in the lungs at 2 dpi and 5 dpi expressed in pmol per gram lung tissue (top panel) or in pmol per mL BALF (bottom panel). Data are presented as mean } sd of n=3 uninfected mice and n=9–10 infected mice per timepoint. Scale Bar=50 μm; U/I=mock infected; dpi=days post-infection; ns=not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 indicate significant differences.

FIGURE 5
FIGURE 5

GPR183 antagonism resulted in less SARS-CoV-2 infection-induced weight loss and in reduced macrophage infiltration. C57BL/6J and Gpr183−/− mice were infected intranasally with approximately 8×104 PFU of mouse-adapted SARS63 CoV-2. Mice were subsequently treated orally with 7.6 mg·kg−1 NIBR189 or vehicle control twice daily from 1 dpi until the end of the experiment. a) Experimental design; b) Weights of mice displayed as percentage of the weight at time of inoculation. c) Representative IHC images of IBA1 in lung of C57BL/6J and Gpr183-/- mice with the respective treatment groups at 2 dpi and 5 dpi (left). Scale Bar=100 μm. Quantitative analysis of IBA1 (right). Data are presented as mean } sd of n=9–12 infected mice per genotype and timepoint. Scale Bar=100 μm; U/I uninfected; dpi=days post-infection; ns=not significant; *, p<0.05; **, p<0.01; ***, p<0.001 indicate significant differences.

GPR183 antagonism reduces macrophage infiltration and inflammatory cytokine expression in the lung of SARS-CoV-2 infected mice

Next, we investigated whether GPR183 antagonism decreases macrophage infiltration and inflammatory cytokines in the lung. SARS-CoV-2-infected C57BL/6J mice treated with NIBR189 had significantly reduced macrophage infiltration into the lung at 2 and 5 dpi (figure 5c). NIBR189 treatment was associated with reduced Tnf, Il10 and Ifng mRNA expression at 2 dpi (figure 6a and Figure S16), and reduced Tnf, Il1b and Il6 expression at 5 dpi (figure 6b and Figure S16). Early interferon responses were not affected by NIBR189 with comparable Ifnb and Ifnl expression at 2 dpi in C57BL/6J mice, but late interferon responses (5 dpi) were significantly lower in NIBR-treated animals compared to controls (figure 6b). No differences between treatment groups were observed for mRNAs encoding Ccl2, Il1b, or Il6 at 2 dpi and Ccl2, Il10 and Ifng at 5 dpi (Figure S16). These results demonstrate that reduced macrophage infiltration in NIBR189-treated mice was associated with reduced pro-inflammatory cytokine expression in the lung, while the early anti-viral IFN responses remained unchanged.

FIGURE 6
FIGURE 6

GPR183 antagonism led to reduced inflammatory cytokine profile. C57BL/6J and Gpr183-/- mice were infected intranasally with approximately 8×104 PFU of mouse-adapted SARS-CoV-2. Mice were subsequently treated orally with 7.6 mg·kg−1 NIBR189 or vehicle control twice daily from 1 dpi until the end of the experiment. Relative expression of (a) Tnf, Ifng, Ifnb and Ifnl at 2 dpi and b) Tnf, Il6, Ifnb and Ifnl at 5 dpi in the lungs measured by RT-qPCR, normalised to Hprt. Data are presented as mean } sd of n=3 uninfected mice and n=9–12 infected mice per genotype and timepoint. U/I=mock infected; dpi=days post-infection; ns=not significant; *, p<0.05; **, p<0.01; ***, p<0.001 indicate significant differences.

GPR183 antagonism reduces SARS-CoV-2 loads

Finally, we investigated whether NIBR189 treatment is associated with altered viral loads. SARS-CoV-2 nucleocapsid protein (Np) expression was reduced in NIBR189-treated C57BL/6J mice compared to those administered vehicle at 2 dpi (figure 7a,b). Np expression was not detected at 5 dpi, when the animals recovered from the infection. However, at the mRNA level, viral Mpro RNA loads in the lungs of NIBR189-treated mice were significantly lower at 5 dpi (figure 7c). Corroborating this, viral PFUs were significantly lower at both 2 and 5 dpi in NIBR-treated animals (Figure S17). In summary, we demonstrate here that GPR183 antagonism reduces viral loads, macrophage infiltration and production of pro-inflammatory cytokines in SARS-CoV-2 infection.

FIGURE 7
FIGURE 7

Mice treated with GPR183 antagonist had lower SARS-CoV-2 loads. C57BL/6J and Gpr183−/− mice were infected intranasally with approximately 8×104 PFU of mouse-adapted SARS-CoV-2. Mice were subsequently treated orally with 7.6 mg·kg−1 NIBR189 or vehicle control twice daily from 1 dpi until the end of the experiment. a) Representative IHC images of viral nucleocapsid (Np) expression at 2 dpi and 5 dpi. b) Quantitative analysis of viral Np expression of the treatment groups at 2 dpi. c) Viral load was assessed in the lung through the detection of Mpro RNA by RT-qPCR at 5 dpi, normalised to HPRT. Data are presented as mean } sd of n=9–12 infected mice per genotype and timepoint. Scale Bar=50 μm; U/I=mock infected; dpi=days post infection; ns=not significant; *, p<0.05; **, p<0.01, indicate significant differences.

Lung macrophages from COVID-19 patients upregulate CH25H, CYP7B1, HSD3B7 and GPR183

To determine whether the oxysterol producing enzymes are increased in humans during SARS-CoV-2 infection we analysed scRNASeq data from healthy controls and COVID-19 patients with moderate and severe disease [12]. We found that CH25H, CYP7B1 and HSD3B7 were significantly upregulated in COVID-19 and almost exclusively expressed in macrophages (figure 8). While GPR183 expression increased significantly in macrophages and myeloid DCs during COVID-19, its expression remained unchanged on other immune cell types.

FIGURE 8
FIGURE 8

Single cell RNASeq expression analysis of cells collected by bronchoalveolar lavage from healthy controls and COVID19 patients. Summary heatmap (left panels) showing average normalised expression level of gene (a) Ch25h (b) Cyp7b1, (c) Hsd3b7 and (d) Gpr183 per individual per cell type cluster. Summary heatmap (right panels) show the average log fold change (logFC) in expression of each gene in 24 cell type clusters between: (1) moderate COVID-19 cases (“M”) and healthy controls (“H”); (2) severe COVID-19 cases (“S”) and healthy controls; (3) COVID-19 cases (“M&S””) and healthy controls; and (4) severe and moderate COVID-19 cases, respectively. The logFC values were estimated using (1) negative binomial generalised linear models applied to raw UMI counts, adjusting for total UMI counts per cell, number of genes detected per cell and percent mitochondrial counts per cell (“NB regression”); or (2) non-parametric Wilcoxon rank sum test applied to normalised counts. Significant associations are highlighted with a single asterisk if they surpass Bonferroni significance (p<1.30×10−4) or a double asterisk if they were further expressed in at least 5% of cells in both groups with an absolute value of logFC greater than 0.25. A logFC greater than zero suggests the expression level of the gene is higher among the focal group (e.g., moderate COVID-19 cases) compared to the other group (e.g., healthy controls), or vice versa.

Discussion

We report that 25-OHC and 7α,25-OHC are produced in the lung upon IAV or SARS-CoV-2 infection attracting monocytes-macrophages in a GPR183-dependent manner. Reduced macrophage infiltration and inflammatory cytokine production in Gpr183−/− mice and NIBR189-treated C57BL/6J mice, significantly improved SARS-CoV-2 infection severity. The antagonist attenuated SARS-CoV-2, but not IAV loads. Whether this is due to pathogen-specific effects or due to more severe disease observed in the IAV model compared to the SARS-CoV-2 model, remains to be investigated. However, macrophage infiltration and inflammatory cytokine production was reduced in both viral models.

In animal models of IAV and SARS-CoV-2 myeloid cells rapidly infiltrate into the lungs [3, 4, 24]. Patients with severe COVID-19 had higher proportions of GPR183+ macrophages and more activated macrophages in BALF [23] strongly implicating macrophages as key contributors to COVID-19-associated hyperinflammation. BALF from severe COVID-19 patients was enriched in the chemokines CCL2 and CCL7 that recruit monocytes to the lung via the chemokine receptor CCR2 [25]. Historically, chemokines have been considered as the main drivers of immune cell migration into the lung; however, our work here reveals that oxysterols have a non-redundant role in monocyte-macrophage infiltration. We further demonstrate that lung macrophages from COVID-19 patients express higher levels of the oxysterol producing enzymes and GPR183 indicating that this mechanism is conserved in humans. Similar to our observations in Gpr183−/− mice, mice lacking CCR2 have delayed macrophage infiltration into the lung [21], however CCR2 is also required for T cell migration. Therefore, animals lacking CCR2 had delayed T cell infiltration and higher viral titres [26]. Although GPR183 is expressed on T cells it is not essential for T cell migration into the lung [27] and antagonising GPR183 did not negatively impact the T cell compartment nor other immune cell subsets.

We recently showed in a murine model of Mycobacterium tuberculosis (Mtb) infection that both GPR183 and the 7α,25-OHC-producing enzyme CYP7B1 are required for rapid macrophage infiltration into the lung upon mycobacterial infection [18]. In the Mtb model, GPR183 was also required for infiltration of eosinophils [14]. We identified both alveolar macrophages and infiltrating macrophages as the predominant cell type expressing CH25H and CYP7B1 upon Mtb infection [18] and corroborated this here with the scRNASeq data from COVID-19 patients.

Deletion of Ch25h has been previously shown to be protective in a mouse model of influenza [28]. 25-OHC was increased in a model of acute lung injury (ALI), however it was decreased in a house dust mite induced model of asthma [8]. Intra-tracheal administration of 25-OHC improved inflammatory markers in the ALI model, whereas it worsened the hallmarks of the asthma model. In both models Gpr183 expression was unaffected. This suggests that 25-OHC production is model specific.

Here we show that reduced macrophage infiltration in Gpr183−/− mice and NIBR189-treated C57BL/6J mice was associated with reduced pro-inflammatory cytokine production, likely due to lower numbers of macrophages present in the tissue. Although we did not observe a NIBR189-mediated reduction in Tnf, Il6 or Ifnb expression in IAV-infected or LPS-stimulated BMDMs (Figure S18), we cannot completely exclude a direct effect of NIBR189 on cytokine production by other immune cells. Irrespective of the exact mechanism, lower pro-inflammatory cytokine production in NIBR189-treated animals may explain, at least in part, the better disease outcomes.

While pro-inflammatory cytokines can be detrimental to the host [30], early type I and III IFNs are crucial in controlling viral replication during IAV [31, 32] and SARS-CoV-2 infection [33, 34]. NIBR189 did not alter early type I or III IFN responses in SARS-CoV-2-infected animals, suggesting that the anti-viral response was not impaired by the treatment.

Several oxysterols can have a direct anti-viral effect [9]. CH25H/25-OHC have been shown to inhibit SARS-CoV-2 infection in vitro by blocking the virus-host cell membrane fusion [36, 37]. Whether NIBR189, which is structurally different from oxysterols, affects viral entry/replication remains to be elucidated.

Other immunosuppressive therapies used in severe COVID-19 like glucocorticoids increase ACE2 expression which promotes viral entry/replication [38, 39] and delays SARS-CoV-2 clearance [40]. NIBR189 did not increase Ace2 mRNA (Figure S19), but Ace2 expression was downregulated to lesser extent in NIBR189-treated animals, which is consistent with lower viral loads.

In summary, we provide the first preclinical evidence of GPR183 as a novel host target for therapeutic intervention to reduce macrophage-mediated hyperinflammation, SARS-CoV-2 loads and COVID-19 severity.

Acknowledgements

This study was supported by grants to KR from the Mater Foundation, the Australian Respiratory Council, Diabetes Australia, the Australian Infectious Diseases Research Centre. SB was supported by an early career seed grant from the Mater Foundation. The Translational Research Institute is supported by a grant from the Australian Government. The Danish Council for Independent Research I Medical Sciences supported MMR. MJS, KRS and JPL are supported by a National Health and Medical Research Council of Australia Investigator grant (1194406), Investigator Grant (2007919) and Senior Research Fellowship (1136130) respectively. We thank the Queensland Health Forensic and Scientific Services, Queensland Department of Health, for providing SARS-CoV-2 isolate. We thank A/Prof Sumaira Hasnain for sharing antibodies used in this study. We acknowledge the technical assistance of the team that operates and maintains the Australian Galaxy service (https://usegalaxy.org.au/). We thank Profs David Hume, Maher Gandhi, and Dr Jake Gratten from the Mater Research Institute – The University of Queensland for critical review of the manuscript.

Footnotes

  • Funder: Australian Infectious Diseases Research Centre; Grant: NA; Australian Respiratory Council; DOI: http://dx.doi.org/10.13039/501100001031; Grant: NA; Danmarks Frie Forskningsfond; DOI: http://dx.doi.org/10.13039/501100004836; Diabetes Australia; DOI: http://dx.doi.org/10.13039/501100000971; Grant: NA; Mater Foundation; DOI: http://dx.doi.org/10.13039/100015471; Grant: NA; National Health and Medical Research Council; DOI: http://dx.doi.org/10.13039/501100000925.

  • Author contributions: Conceptualization: CXF, SB, MJS, KRS, MMR, KR Methodology: KYC, HBO, BJA, BM, SR Investigation: CXF, SB, KYC, MDN, HBO, BJA, BM, SR, RW, CS, LB, KB, SS, JES, RP, YY, JPL AK Writing-original draft: CXF, SB, KR Writing-review and editing: all authors. Funding acquisition: SB, KRS, MMR, KR.

  • Conflict of interest: Stacey Bartlett reports an early career seed grant from the Mater Foundation, supporting the present study.

  • Conflict of interest: Helle Bielefeldt-Ohmann reports consulting fees from Paradigm Biopharma, Queensland Univ. Technol., Colorado State University; outside the submitted work.

  • Conflict of interest: Matthew J. Sweet reports grants from National Health and Medical Research Council of Australia; outside the submitted work.

  • Conflict of interest: Kavita Bisht reports grants from American Society of Hematology (ASH) Global Research Award., Translational Research Institute-Mater Research LINC grant, Mater foundation; outside the submitted work.

  • Conflict of interest: Yuanhao Yang reports grants from Mater Foundation, supporting the present study.

  • Conflict of interest: Jean-Pierre Levesque reports grants from National Health and Medical Research Council, US Department of Defense; royalties or licences from GlycoMimetics Inc.; outside the submitted work.

  • Conflict of interest: Mette Marie Rosenkilde reports support for the animal studies and breeding in Denmark of mouse strain used in this study from Independent Research Fund Denmark; grants from Independent Research Fund Denmark, Novo Nordisk Foundation, Donation from deceased Valter Alex Torbjørn Eichmuller (VAT Eichmuller)-2020-117043, Kirsten and Freddy Johansens Foundation (KFJ) - 2017-112697; royalties from Antag Therapeutics and Bainan Biotech from patents made at the UCPH; travel support from Gordon Research Conference 2022; and is the co-founder of the following biotech companies: Antag Therapeutics, Bainan Biotech, Synklino; outside the submitted work.

  • Conflict of interest: Kirsty R. Short reports grants from National Health and Medical Research Council of Australia; consulting fees from Sanofi, Novo Nordisk, Roche; outside the submitted work.

  • Conflict of interest: Katharina Ronacher reports support for the present manuscript from Mater Foundation, Diabetes Australia, Australian Infectious Diseases Research Centre, Australian Respiratory Council; grants from NIH R01 (5R01AI116039); outside the submitted work.

  • Conflict of interest: All other authors have nothing to disclose.

  • Received June 28, 2022.
  • Accepted October 20, 2022.
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References

  1. ↵
    1. Flerlage T,
    2. Boyd DF,
    3. Meliopoulos V, et al.
    Influenza virus and SARS-CoV-2: pathogenesis and host responses in the respiratory tract. Nat Rev Microbiol 2021; 19: 425–441. doi:10.1038/s41579-021-00542-7
    OpenUrlCrossRefPubMed
  2. ↵
    1. Chen ST,
    2. Park MD,
    3. Del Valle DM, et al.
    Shift of lung macrophage composition is associated with COVID-19 disease severity and recovery. bioRxiv 2022.
  3. ↵
    1. Speranza E,
    2. Williamson BN,
    3. Feldmann F, et al.
    Single-cell RNA sequencing reveals SARS-CoV-2 infection dynamics in lungs of African green monkeys. Sci Transl Med 2021; 13: eabe8146. doi:10.1126/scitranslmed.abe8146
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Singh DK,
    2. Aladyeva E,
    3. Das S, et al.
    Myeloid cell interferon responses correlate with clearance of SARS-CoV-2. Nat Commun 2022; 13: 679. doi:10.1038/s41467-022-28315-7
    OpenUrl
  5. ↵
    1. Shen ZJ,
    2. Hu J,
    3. Kashi VP, et al.
    Epstein-Barr virus-induced gene 2 mediates allergen-induced leukocyte migration into airways. Am J Respir Crit Care Med 2017; 195: 1576–1585. doi:10.1164/rccm.201608-1580OC
    OpenUrl
  6. ↵
    1. Jia J,
    2. Conlon TM,
    3. Sarker RS, et al.
    Cholesterol metabolism promotes B-cell positioning during immune pathogenesis of chronic obstructive pulmonary disease. EMBO Mol Med 2018; 10: e8349.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Sugiura H,
    2. Koarai A,
    3. Ichikawa T, et al.
    Increased 25-hydroxycholesterol concentrations in the lungs of patients with chronic obstructive pulmonary disease. Respirology 2012; 17: 533–540. doi:10.1111/j.1440-1843.2012.02136.x
    OpenUrlCrossRefPubMed
  8. ↵
    1. Bottemanne P,
    2. Paquot A,
    3. Ameraoui H, et al.
    25-Hydroxycholesterol metabolism is altered by lung inflammation, and its local administration modulates lung inflammation in mice. FASEB J 2021; 35: e21514. doi:10.1096/fj.202002555R
    OpenUrl
  9. ↵
    1. Foo CX,
    2. Bartlett S,
    3. Ronacher K
    . Oxysterols in the immune response to bacterial and viral infections. Cells 2022; 11: 201. doi:10.3390/cells11020201
    OpenUrl
  10. ↵
    1. Mutemberezi V,
    2. Guillemot-Legris O,
    3. Muccioli GG
    . Oxysterols: from cholesterol metabolites to key mediators. Prog Lipid Res 2016; 64: 152–169. doi:10.1016/j.plipres.2016.09.002
    OpenUrlCrossRefPubMed
  11. ↵
    1. Hannedouche S,
    2. Zhang J,
    3. Yi T, et al.
    Oxysterols direct immune cell migration via EBI2. Nature 2011; 475: 524-527. doi:10.1038/nature10280
    OpenUrlCrossRefPubMedWeb of Science
  12. ↵
    1. Liu C,
    2. Yang XV,
    3. Wu J, et al.
    Oxysterols direct B-cell migration through EBI2. Nature 2011; 475: 519–523. doi:10.1038/nature10226
    OpenUrlCrossRefPubMedWeb of Science
  13. ↵
    1. Emgard J,
    2. Kammoun H,
    3. Garcia-Cassani B, et al.
    Oxysterol Sensing through the Receptor GPR183 Promotes the Lymphoid-Tissue-Inducing Function of Innate Lymphoid Cells and Colonic Inflammation. Immunity 2018; 48: 120–132.e128. doi:10.1016/j.immuni.2017.11.020
    OpenUrl
  14. ↵
    1. Bohrer AC,
    2. Castro E,
    3. Tocheny CE, et al.
    Rapid GPR183-mediated recruitment of eosinophils to the lung after Mycobacterium tuberculosis infection. 2022. 2022.2002.2018.480919.
  15. ↵
    1. Spann NJ,
    2. Glass CK
    . Sterols and oxysterols in immune cell function. Nat Immunol 2013; 14: 893–900. doi:10.1038/ni.2681
    OpenUrlCrossRefPubMed
  16. ↵
    1. Preuss I,
    2. Ludwig MG,
    3. Baumgarten B, et al.
    Transcriptional regulation and functional characterization of the oxysterol/EBI2 system in primary human macrophages. Biochem Biophys Res Commun 2014; 446: 663–668. doi:10.1016/j.bbrc.2014.01.069
    OpenUrl
  17. ↵
    1. Gessier F,
    2. Preuss I,
    3. Yin H, et al.
    Identification and characterization of small molecule modulators of the Epstein-Barr virus-induced gene 2 (EBI2) receptor. J Med Chem 2014; 57: 3358–3368. doi:10.1021/jm4019355
    OpenUrlCrossRef
  18. ↵
    1. Ngo MD,
    2. Bartlett S,
    3. Bielefeldt-Ohmann H, et al.
    A blunted GPR183/oxysterol axis during dysglycemia results in delayed recruitment of macrophages to the lung during M. tuberculosis infection. J Infect Dis 2022; 225: 2219–2228. doi:10.1093/infdis/jiac102
    OpenUrl
  19. ↵
    1. Bartlett S,
    2. Gemiarto AT,
    3. Ngo MD, et al.
    GPR183 Regulates Interferons, Autophagy, and Bacterial Growth During Mycobacterium tuberculosis Infection and Is Associated With TB Disease Severity. Front Immunol 2020; 11: 601534. doi:10.3389/fimmu.2020.601534
    OpenUrl
  20. ↵
    1. Pereira JP,
    2. Kelly LM,
    3. Xu Y, et al.
    EBI2 mediates B cell segregation between the outer and centre follicle. Nature 2009; 460: 1122–1126. doi:10.1038/nature08226
    OpenUrlCrossRefPubMedWeb of Science
  21. ↵
    1. Poczobutt JM,
    2. Gijon M,
    3. Amin J, et al.
    Eicosanoid profiling in an orthotopic model of lung cancer progression by mass spectrometry demonstrates selective production of leukotrienes by inflammatory cells of the microenvironment. PLoS One 2013; 8: e79633. doi:10.1371/journal.pone.0079633
    OpenUrlCrossRefPubMed
  22. ↵
    1. Zhou JH,
    2. Wang YN,
    3. Chang QY, et al.
    Type III Interferons in Viral Infection and Antiviral Immunity. Cell Physiol Biochem 2018; 51: 173–185. doi:10.1159/000495172
    OpenUrlCrossRefPubMed
  23. ↵
    1. Liao M,
    2. Liu Y,
    3. Yuan J, et al.
    Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19. Nat Med 2020; 26: 842–844. doi:10.1038/s41591-020-0901-9
    OpenUrlCrossRefPubMed
  24. ↵
    1. Corry J,
    2. Kettenburg G,
    3. Upadhyay AA, et al.
    Infiltration of inflammatory macrophages and neutrophils and widespread pyroptosis in lung drive influenza lethality in nonhuman primates. PLoS Pathog 2022; 18: e1010395. doi:10.1371/journal.ppat.1010395
    OpenUrl
  25. ↵
    1. Zhou Z,
    2. Ren L,
    3. Zhang L, et al.
    Heightened Innate Immune Responses in the Respiratory Tract of COVID-19 Patients. Cell Host Microbe 2020; 27: 883–890.e882. doi:10.1016/j.chom.2020.04.017
    OpenUrl
  26. ↵
    1. Dawson TC,
    2. Beck MA,
    3. Kuziel WA, et al.
    Contrasting effects of CCR5 and CCR2 deficiency in the pulmonary inflammatory response to influenza A virus. Am J Pathol 2000; 156: 1951–1959. doi:10.1016/S0002-9440(10)65068-7
    OpenUrlCrossRefPubMedWeb of Science
  27. ↵
    1. Hoft SG,
    2. Sallin MA,
    3. Kauffman KD, et al.
    The Rate of CD4 T cell entry into the lungs during mycobacterium tuberculosis infection is determined by partial and opposing effects of multiple chemokine receptors. Infect Immun 2019; 87: e00491-19.
    OpenUrlFREE Full Text
  28. ↵
    1. Gold ES,
    2. Diercks AH,
    3. Podolsky I, et al.
    25-Hydroxycholesterol acts as an amplifier of inflammatory signaling. Proc Natl Acad Sci USA 2014; 111: 10666–10671. doi:10.1073/pnas.1404271111
    OpenUrlAbstract/FREE Full Text
    1. Del Valle DM,
    2. Kim-Schulze S,
    3. Huang HH, et al.
    An inflammatory cytokine signature predicts COVID-19 severity and survival. Nat Med 2020; 26: 1636–1643. doi:10.1038/s41591-020-1051-9
    OpenUrlCrossRefPubMed
  30. ↵
    1. Diamond MS,
    2. Kanneganti TD
    . Innate immunity: the first line of defense against SARS-CoV-2. Nat Immunol 2022; 23: 165–176. doi:10.1038/s41590-021-01091-0
    OpenUrlCrossRefPubMed
  31. ↵
    1. Stanifer ML,
    2. Guo C,
    3. Doldan P, et al.
    Importance of Type I and III Interferons at Respiratory and Intestinal Barrier Surfaces. Front Immunol 2020; 11: 608645. doi:10.3389/fimmu.2020.608645
    OpenUrlCrossRef
  32. ↵
    1. Galani IE,
    2. Triantafyllia V,
    3. Eleminiadou EE, et al.
    Interferon-lambda Mediates Non-redundant Front-Line Antiviral Protection against Influenza Virus Infection without Compromising Host Fitness. Immunity 2017; 46: 875–890.e876. doi:10.1016/j.immuni.2017.04.025
    OpenUrlCrossRefPubMed
  33. ↵
    1. Lokugamage KG,
    2. Hage A,
    3. de Vries M, et al.
    Type I interferon susceptibility distinguishes SARS-CoV-2 from SARS-CoV. bioRxiv 2020.
  34. ↵
    1. Vanderheiden A,
    2. Ralfs P,
    3. Chirkova T, et al.
    Type I and Type III Interferons Restrict SARS-CoV-2 Infection of Human Airway Epithelial Cultures. J Virol 2020; 94: e00985-20. doi:10.1128/JVI.00985-20
    OpenUrlAbstract/FREE Full Text
    1. Cameron MJ,
    2. Ran L,
    3. Xu L, et al.
    Interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome. J Virol 2007; 81: 8692–8706. doi:10.1128/JVI.00527-07
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Wang S,
    2. Li W,
    3. Hui H, et al.
    Cholesterol 25-Hydroxylase inhibits SARS-CoV-2 and other coronaviruses by depleting membrane cholesterol. EMBO J 2020; 39: e106057. doi:10.15252/embj.2020106057
    OpenUrlCrossRefPubMed
  37. ↵
    1. Zang R,
    2. Case JB,
    3. Yutuc E, et al.
    Cholesterol 25-hydroxylase suppresses SARS-CoV-2 replication by blocking membrane fusion. Proc Natl Acad Sci U S A 2020; 117: 32105–32113. doi:10.1073/pnas.2012197117
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Sinha S,
    2. Cheng K,
    3. Schaffer AA, et al.
    In vitro and in vivo identification of clinically approved drugs that modify ACE2 expression. Mol Syst Biol 2020; 16: e9628. doi:10.15252/msb.20209628
    OpenUrlCrossRef
  39. ↵
    1. Saheb Sharif-Askari N,
    2. Saheb Sharif-Askari F,
    3. Alabed M, et al.
    Effect of Common Medications on the Expression of SARS-CoV-2 Entry Receptors in Kidney Tissue. Clin Transl Sci 2020; 13: 1048–1054. doi:10.1111/cts.12862
    OpenUrl
  40. ↵
    1. Li J,
    2. Liao X,
    3. Zhou Y, et al.
    Association between glucocorticoids treatment and viral clearance delay in patients with COVID-19: a systematic review and meta-analysis. BMC Infect Dis 2021; 21: 1063. doi:10.1186/s12879-021-06548-z
    OpenUrl
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GPR183 antagonism reduces macrophage infiltration in influenza and SARS-CoV-2 infection
Cheng Xiang Foo, Stacey Bartlett, Keng Yih Chew, Minh Dao Ngo, Helle Bielefeldt-Ohmann, Buddhika Jayakody Arachchige, Benjamin Matthews, Sarah Reed, Ran Wang, Christian Smith, Matthew J. Sweet, Lucy Burr, Kavita Bisht, Svetlana Shatunova, Jane E. Sinclair, Rhys Parry, Yuanhao Yang, Jean-Pierre Lévesque, Alexander Khromykh, Mette Marie Rosenkilde, Kirsty R. Short, Katharina Ronacher
European Respiratory Journal Jan 2022, 2201306; DOI: 10.1183/13993003.01306-2022
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