Monocyte-macrophage membrane expression of IL-1R2 is a severity biomarker in sepsis

Categories: Disease & Virus

April 10, 2025

Characteristics of the population

Patients analyzed in the present study were part of a population of 178 subjects admitted to Humanitas Research Hospital [12]. In this cohort, a total of 117 patients were analyzed based on the availability of PBMCs. Patients were subdivided into non-sepsis (n = 23), sepsis (n = 69), and septic shock (n = 25) in accordance with the Sepsis-3 Consensus Definition of Sepsis [1]. 19 age-matched healthy controls were enrolled. Table S2 summarizes the demographic and clinical characteristics of patient cohorts with the exception of two non-sepsis individuals whose clinical data were not available. The median (Q1-Q3) age was 76 (68–84) years, and the percentage of male subjects (64%) was higher compared to females (36%). Median age of healthy controls was 58 years (54–64), 7 were males (37%), and 12 were females (63%).

Most of the sepsis patients were diagnosed with a respiratory origin infection (n = 31, 45%), while the urinary tract (n = 11, 44%) was the most common infection site in septic shock patients. Creatinine, urea, potassium levels, SOFA, and qSOFA scores increased from non-sepsis to sepsis and septic shock patients.

Immunophenotyping of PBMCs in sepsis

PBMCs were analyzed by multiparametric flow cytometry analysis. The frequency of CD3⁺ T lymphocytes was very heterogeneous in patients and lower in non-sepsis and sepsis patients compared to healthy individuals, in agreement with relative lymphopenia in sepsis [4] (Fig. S2a). The reduction of T lymphocytes did not reach statistical significance in septic shock patients compared to healthy donors (Fig. S2a). The frequency of both conventional CD4⁺ T cells (Tconv) and regulatory T cells (Treg) was reduced in all patient groups (Fig. S2b, c). Conversely, CD8⁺ T cells, B lymphocytes, and NK cells (Fig. S2d–f) were not significantly affected. The CD4/CD8 ratio, a prognostic risk factor in infections, was reduced in all infected patients compared to healthy controls, but it was not associated with disease severity (Fig. S2g).

As expected [5], the frequency of circulating monocytes, although very heterogeneous in patients, was higher in infected individuals (Fig. 1a), contributing to a lower CD3⁺/Monocytes ratio (Fig. S2h). Monocytes were further dissected into CD14⁺CD16⁻ classical monocytes, CD14⁺CD16⁺ intermediate monocytes, and CD14^–CD16⁺ non-classical monocytes [18]. Increased frequency of CD14⁺CD16⁺ monocytes and reduced frequency of CD14⁺CD16⁻ monocytes was observed in septic and septic shock patients compared to healthy controls (Fig. 1b). In line with other studies [5, 13, 19, 20] HLA-DR was downregulated in monocytes of severely infected individuals compared to healthy donors, and was further reduced in sepsis and septic shock patients, negatively associating with the disease severity (Fig. 1c). In agreement, HLA-DR expression in monocytes was negatively correlated with the SOFA score, especially in non-sepsis and sepsis cohorts (Spearman r = −0.27, p = 0.012) (Fig. 1d, e and Table 1). When all the infected patients were considered, this correlation was lost (Spearman r = −0.16, p = 0.11, Table 1), due to the high heterogeneity observed in patients with SOFA score > 10.

**Fig. 1: IL-1R2 expression and characterization of leukocytes in septic conditions.**

Table 1 Correlation of IL-1R2, MS4A4A, and HLA-DR with clinical parameters.

These data indicate that sepsis was associated with lymphopenia and monocytosis and that our cohort of patients met sepsis-associated immunopathological criteria. Downregulation of HLA-DR and increased frequency of CD14⁺CD16⁺ subset indicated that monocytes were not only quantitatively but also qualitatively affected by the disease severity.

Expression of IL-1R2 in PBMCs in sepsis

We previously showed that plasma concentration of sIL-1R2 increases in patients with sepsis and septic shock, correlating with severity and mortality [12]. To address whether the membrane-IL-1R2 was also modulated in sepsis, its expression was evaluated by flow cytometry.

IL-1R2 was mostly expressed by monocytes, whereas lymphoid cells showed low levels of the protein in healthy conditions, which was upregulated reaching the highest levels in septic patients (Fig. 1f–m). The induction of IL-1R2 observed in CD3⁺ T cells from septic patients (2.3-fold increase compared to healthy donors) (Fig. 1f), was observed in all T cell subsets, including CD4⁺ (Fig. 1g, h) and CD8⁺ T cells (Fig. 1i). Increased expression of IL-1R2 was observed in B cells (1.8-fold increase) and NK cells (2.8-fold increase) from septic patients (Fig. 1j, k).

In monocytes, IL-1R2 was mostly upregulated in the sepsis cohort (3.96-fold increase) (Fig. 1l).

CD14⁺CD16⁻ and CD14⁺CD16⁺ monocyte subsets expressed the highest levels of IL-1R2 in healthy donors compared to non-classical monocytes CD14^–CD16⁺ (Fig. 1m). In sepsis patients CD14⁺CD16⁻, CD14⁺CD16⁺, and CD14⁻CD16⁺ cells showed, respectively, 3.8-, 2.7-, and 2.5-fold increase of IL-1R2 MFI, compared to healthy subjects (Fig. 1m).

In both monocytes and lymphocytes, IL-1R2 cell surface expression was reduced in septic shock, compared to sepsis (Fig. 1f–m). Thus, further statistical analyzes were also performed, excluding septic shock patients.

Collectively, multiparametric flow cytometry analysis revealed upregulation of IL-1R2 on the plasma membrane, with the highest levels in sepsis.

IL-1R2 is transcriptionally regulated during monocyte-to-macrophage differentiation

To better investigate IL-1R2 regulation, we focused on monocytes, taking advantage of a single-cell RNA sequencing (scRNA-seq) study of septic patients with urinary tract infections [13]. IL-1R2 was shown to be overexpressed by a cluster of IL-1R2⁺ HLA-DR^low monocytes (named MS1) emerging in the bloodstream as a result of sepsis-induced dysregulated myelopoiesis [13]. In particular, Differentially Expressed Genes (DEGs) were identified by comparing IL-1R2⁺ HLA-DR^low MS1 cluster vs All Monocytes (Fig. 2a) and vs the IL-1R2^low HLA-DR⁺ MS2 cluster (Fig. 2b). Having observed genes typically associated with macrophages (e.g., MS4A4A, CD63, CD163) [21,22,23,24] in the MS1 cluster, we further investigated potential differentiation of monocytes to macrophages in sepsis. We evaluated the expression of MS1 DEGs in monocytes and in macrophages using a publicly available dataset from the Human Protein Atlas. The expression was extrapolated as Normalized transcript expression values, or nTPM, and compared using the formula nTPM of Macrophages—nTPMs of Monocytes/nTPMs of Macrophages + nTPMs of Monocytes, which allowed us to represent transcripts with very heterogeneous expression on a relative scale from −1 to 1 (Fig. 2a, b).

**Fig. 2: Association of IL-1R2 to Mono/Mφ functional signatures in monocytic cells in sepsis.**

Intriguingly, IL-1R2 was mainly expressed by macrophages (Fig. 2a, b), suggesting that it is associated with myeloid cell differentiation. Moreover, most of MS1 signature genes (> 55.68% in Fig. 2a, and >63.6% in Fig. 2b) were highly expressed by macrophages. Among the DEGs (Fig. 2a, b), we focused on MS4A4A, a tetraspan molecule associated with anti-inflammatory markers and involved in Dectin-1-dependent activation of macrophages [21]. The molecule is poorly expressed by monocytes and upregulated following differentiation into macrophages [22]. We also found CD63, a tetraspanin family member expressed by monocyte-derived macrophages [23] and negatively associated with survival of sepsis patients [25, 26]. In agreement with transcriptional data [13, 27], flow cytometry analysis revealed a significant upregulation of both CD63 and MS4A4A in monocytes from our cohort of patients (Fig. 2c, d). Specifically, MS4A4A expression was upregulated in monocytes of sepsis patients (Fig. 2e), and increased levels of CD63 were found in the septic shock cohort, despite the limited number of patients analyzed for this marker.

HLA-DR downregulation in monocytes has been associated with myeloid cell dysfunction and correlated with the SOFA score [19, 20], as confirmed in our non-sepsis plus sepsis cohort (Fig. 1d, e and Table 1). To better understand the association between IL-1R2 expression and the functional state of monocytes in sepsis, we investigated the correlation between IL-1R2 and HLA-DR levels in monocytic cells. HLA-DR was negatively correlated with IL-1R2 (Spearman r = −0.48, p < 0.0001) (Table 1). In parallel, we observed a negative correlation of MS4A4A, used here as marker of differentiation, with HLA-DR (Spearman r = −0.26, p = 0.004) (Table 1). A weak but significant correlation of IL-1R2 with MS4A4A was found by evaluating all individuals enrolled in the study (Spearman r = 0.22, p = 0.015), whereas a strong correlation was observed in healthy donors and non-sepsis patients (Spearman r = 0.61, p < 0.0001), suggesting rapid and concomitant induction of the two molecules upon infection.

Collectively, these results suggest that the upregulation of IL-1R2 was associated with the acquisition of monocyte-to-macrophage differentiation markers (e.g., MS4A4A) and downregulation of HLA-DR.

Regulation of IL-1R2 by CSFs and lipopolysaccharide

The differentiation of monocytes to macrophages is promoted by M-CSF and GM-CSF [28, 29], and increased levels of circulating CSFs were reported in sepsis [30].

To experimentally mimic the phenotypic transition from monocyte to macrophage, we investigated the phenotype of monocytes stimulated with M-CSF or GM-CSF for a time shorter than usual, e.g., 4 instead of 7 days, and in the presence of lipopolysaccharide (LPS). As shown in Fig. 2f, h, stimulation of monocytes with M- or GM-CSF was associated with the induction of IL-1R2, CD63, and MS4A4A. M-CSF stimulated cells expressed significantly higher levels of these proteins than GM-CSF stimulated cells (Fig. 2f–h). CD14⁺ HLA-DR⁺ cells were predominant after M-CSF stimulation (Fig. 2i bottom panels, 2j), and no significant changes in cluster frequency were observed after stimulation with LPS (Fig. 2j). In contrast, GM-CSF monocytic cells resulted in three distinct Mono/Mφ clusters, namely HLA-DR⁺CD14^lowCD16⁻ (or HLA-DR⁺, in green), HLA-DR^–CD14⁺CD16⁻ (or CD14⁺CD16⁻, in blue), and HLA-DR^–CD14⁺CD16⁺ (or CD14⁺CD16⁺, in red) (Fig. 2i upper panels, Fig. 2k). The stimulation with LPS promoted the reduction of HLA-DR⁺ cell frequency (Fig. 2k), as observed in septic patients.

In addition, in line with previous studies [31], the activation of M-CSF-generated macrophages with LPS was associated with downregulation of IL-1R2 (Fig. 2l). In contrast, the stimulation with LPS of GM-CSF-generated macrophages triggered the upregulation of IL-1R2 (Fig. 2m). As reported in sepsis, IL-1R2 expression in HLA-DR⁺ monocytic cells was lower upon stimulation with LPS (Fig. 2m). These results indicated that LPS has a divergent effect on IL-1R2 in GM-CSF- and M-CSF-induced Mono/Mφ. Combination of GM-CSF and LPS recapitulated the reduction of HLA-DR, the upregulation of IL-1R2, and other markers of monocyte-to-macrophage transition as observed in patients with sepsis.

To investigate the molecular mechanisms involved in IL-1R2 regulation, we took advantage of publicly available ChIP-seq data of monocytes and macrophages, finding a specific enrichment of the myeloid-specific transcription factors CEBPB and SPI1 on IL1R2 in macrophages (Fig. S3). Moreover, recruitment of POLII and reduction of the inhibitory epigenetic modification H3K4me27 were observed (Fig. S3). Of note, increased accessibility of CEBPB and SPI1 binding sites was reported in monocytes from patients with sepsis [13]. These results suggest that CEBPB and SPI1 are involved in the regulation of IL1R2 expression in macrophages, as well as in monocytes from patients with sepsis.

To understand the biological significance of IL-1R2, we investigated the association between IL-1R2 and other functional markers of polarization, such as arginase-1 (ARG-1), IL-10, CD204, PD-L1, and SIRPα. As shown in Fig. 3a, monocytic cells differently expressed IL-1R2, but we did not identify separate IL-1R2⁺ and IL-1R2⁻ clusters. We thus compared the first quartile (Q1) expressing low levels of IL-1R2 (IL-1R2^low) and the fourth quartile (Q4) expressing high levels of IL-1R2 (IL-1R2^high) (Fig. 3a). ARG-1 (Fig. 3b) and IL-10 (Fig. 3c) expression was higher in IL-1R2^high compared to IL-1R2^low, indicating a positive association between these immune suppressive molecules and IL-1R2. In addition, the differentiation marker and scavenger receptor CD204 (Fig. 3d) and the immune checkpoint (IC) PD-L1 (Fig. 3e) were upregulated in the IL-1R2^high subset. No differences were found between IL-1R2^high and IL-1R2^low on SIRPα, an IC specifically induced by GM-CSF but not by M-CSF (Fig. 3f). Interestingly, SIRPα was included among the scRNA-sequencing signature genes (FDR = 0.01) of sepsis monocytes [13].

**Fig. 3: Markers of human IL-1R2^high Mono/Mφ cells and clinical significance.**

Collectively, these results indicate that IL-1R2 is part of a signature associated with Mono/Mφ differentiation (e.g., MS4A4A upregulation) preferentially in response to CSFs and LPS, including immunosuppressive markers, such as ARG-1, IL-10, PD-L1, and CD204.

Membrane-associated IL-1R2 as a marker of sepsis

We next assessed the clinical significance of membrane-IL-1R2 in PBMCs. Progressively increasing monocyte IL-1R2 expression was observed in patients with SOFA score < 7 (Fig. 4a), reaching a plateau at this level of severity. High heterogeneity was observed in patients with SOFA score > 10. Monocyte IL-1R2 correlated with SOFA score especially in non-sepsis and sepsis cohorts (Spearman r = 0.47, p < 0.0001), faring more pronounced than HLA-DR as a biomarker of severity (Table 1). Statistically significant positive correlation was also observed in T and B lymphocytes from non-sepsis and sepsis cohorts (Table S3). No major differences were found stratifying patients by the site of infection origin and antibiotic treatment, as well as corticosteroid treatment, which is known to regulate IL-1R2 [32, 33] (Fig. S4). In addition, monocyte IL-1R2 significantly correlated with urea and creatinine (Table 1).

**Fig. 4: Association of monocyte membrane-IL-1R2 with mortality.**

We next examined the association of IL-1R2 with pro-inflammatory cytokines and soluble mediators belonging to sepsis-associated cytokine storm [2, 12, 34]. The soluble and membrane-associated IL-1R2 were concomitantly upregulated (Fig. S5a), and positively correlated in non-sepsis and septic individuals (Table S4). Membrane-IL-1R2 expression was lower in monocytes of septic shock patients compared to sepsis, whereas sIL-1R2 plasma levels were higher [12], suggesting release from the cell membrane. However, we did not observe a negative correlation between the two forms in this cohort (Spearman r = 0.05, p = 0.63), suggesting that sIL-1R2 may be released from other cell types in addition to monocytes. IL-1R2 positively correlated with IL-6, IL-10, IL-8, and PTX3 [12] in non-sepsis and sepsis patients (Fig. S5b–i and Table S4).

To better elucidate the regulation of membrane-IL-1R2 in PBMCs in sepsis, we analyzed its expression by qPCR. MS4A4A and CD14 were included as monocyte/macrophage reference genes. We observed a trend of increase in IL1R2 mRNA levels depending on the severity, including in septic shock (Fig. S6a). The same trend was observed for MS4A4A (increase) and CD14 (decrease) according to severity (Fig. S6b, c), and in agreement with flow cytometry results (Fig. 1). Collectively, these results suggest that post-transcriptional regulation and shedding of IL-1R2 are responsible for the downregulation of membrane-IL-1R2 expression and the increase of sIL-1R2 plasma levels.

Since MS4A4A was upregulated in patients with sepsis and correlated with IL-1R2 and HLA-DR, we evaluated its clinical significance. MS4A4A expression increased with the SOFA score as well (Fig. S7a), reaching its plateau at SOFA score 10. As observed for HLA-DR (Fig. 1e, f) and IL-1R2 (Fig. 4a), patients with SOFA score > 10 showed heterogeneous expression of MS4A4A (Fig. S7a). In agreement, MS4A4A positively correlated with the SOFA score in non-sepsis and sepsis subgroups (Spearman r = 0.3, p = 0.004), but not in the entire cohort (Table 1). Moreover, MS4A4A positively correlated with pro-inflammatory cytokines and sepsis-associated factors in the entire cohort (Table S4).

The clinical significance of IL-1R2 expression was investigated by stratifying patients according to outcome within 90 days of hospitalization. Membrane-IL-1R2 showed a significant reduction in septic shock patients with adverse outcome (Fig. 4b). Based on these results, we specifically investigated the correlation between IL-1R2, pro-inflammatory cytokines, and soluble factors in septic shock. Membrane-associated IL-1R2, but not sIL-1R2, negatively correlated with the IL-1 family members IL-1β and IL-18 (Table S5).

Septic shock patients with adverse outcome showed high levels of PTX3, among the inflammatory markers tested, in line with our previous study on the entire cohort [12] (Fig. S7b). Having found a correlation between membrane-IL-1R2 and soluble factors, Cox proportional hazards models were used after adjusting for IL-1-related biomarkers (i.e., sIL-1R2, IL-1β, IL-18, IL-1ra) and PTX3. IL-1R2 was not associated with survival in either the non-sepsis or sepsis cohorts (HR = 0.84, 95% CI: 0.10–6.77, p = 0.87). In contrast, a strong inverse association with survival emerged in patients with septic shock (HR = 0.02, 95% CI: 0.00–1.01, p = 0.05). These findings were further supported by proportional hazards additive models, which revealed a non-linear contribution of IL-1R2 to mortality in septic shock, but not in non-sepsis and sepsis cohorts (Fig. 4c, d).

Finally, we evaluated whether monocyte IL-1R2 expression stratified patients through a ROC curve analysis. IL-1R2 emerged as an accurate marker of disease discriminating healthy donors vs non-sepsis and sepsis subgroups, and stratifying non-sepsis and sepsis patients admitted to the emergency department (Fig. 4e). By comparison, the ROC curve analysis of MS4A4A expression showed that this marker discriminated healthy donors vs non-sepsis or sepsis groups (p < 0.0001), but did not stratify non-sepsis and sepsis patients (Fig. S7c).

Collectively, these results indicate that membrane-IL-1R2 on monocytic cells associates with markers of macrophage differentiation and dysfunction in sepsis. IL-1R2 expression positively correlates with the SOFA score and discriminates non-sepsis from sepsis individuals, whereas in septic shock, IL-1R2 is downregulated due to post-translational regulation, and it serves as a prognostic marker.

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