PGLYRP1-mIgG2a-Fc inhibits macrophage activation via AKT/NF-κB signaling and protects against fatal lung injury during bacterial infection

Yan Jia; Shan Ren; Luyao Song; Siyi Wang; Wei Han; Jingjing Li; Yan Yu; BuYong Ma

doi:10.1016/j.isci.2023.106653

. 2023 Apr 14;26(5):106653. doi: 10.1016/j.isci.2023.106653

PGLYRP1-mIgG2a-Fc inhibits macrophage activation via AKT/NF-κB signaling and protects against fatal lung injury during bacterial infection

Yan Jia ¹, Shan Ren ², Luyao Song ¹, Siyi Wang ¹, Wei Han ¹, Jingjing Li ^1,^3,^∗, Yan Yu ^2,^∗∗, BuYong Ma ^1,^∗∗∗

PMCID: PMC10102533 PMID: 37113764

Summary

Severe bacterial pneumonia leads to acute respiratory distress syndrome (ARDS), with a high incidence rate and mortality. It is well-known that continuous and dysregulated macrophage activation is vital for aggravating the progression of pneumonia. Here, we designed and produced an antibody-like molecule, peptidoglycan recognition protein 1-mIgG2a-Fc (PGLYRP1-Fc). PGLYRP1 was fused to the Fc region of mouse IgG2a with high binding to macrophages. We demonstrated that PGLYRP1-Fc ameliorated lung injury and inflammation in ARDS, without affecting bacterial clearance. Besides, PGLYRP1-Fc reduced AKT/nuclear factor kappa-B (NF-κB) activation via the Fc segment bound Fc gamma receptor (FcγR)-dependent mechanism, making macrophage unresponsive, and immediately suppressed proinflammatory response upon bacteria or lipopolysaccharide (LPS) stimulus in turn. These results confirm that PGLYRP1-Fc protects against ARDS by promoting host tolerance with reduced inflammatory response and tissue damage, irrespective of the host’s pathogen burden, and provide a promising therapeutic strategy for bacterial infection.

Subject areas: Biochemistry, Molecular biology, Immunology

Graphical abstract

Highlights

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A specific inhibitor (PGLYRP1-Fc) was developed for macrophage activation
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FcγR targeted by PGLYRP1-Fc was an effective strategy for protecting against ARDS
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PGLYRP1-Fc improved lung injury and reduced the mortality of ARDS mice PGLYRP1-Fc reduced inflammation via inhibiting FcγR-dependent AKT/NF-κB activation

Biochemistry; Molecular biology; Immunology

Introduction

Acute respiratory distress syndrome (ARDS) is a common cause of respiratory failure in critically ill patients and is defined by the acute onset of noncardiogenic pulmonary edema, hypoxemia, and the need for mechanical ventilation.¹ ARDS occurs most often in pneumonia, sepsis, aspiration of gastric contents, or severe trauma and is present in the high incidence rate and mortality.² The current care for ARDS is symptomatic treatment, such as adopting microbial killing and/or neutralization, plus supportive care and mechanical ventilation.³ Bacterial pneumonia stands out as the most common cause of ARDS-associated deaths. The therapeutic intervention used for bacterial pneumonia is antibiotics, which often cause bacterial resistance.⁴ Unfortunately, there is no effective drug to reduce the short-term or long-term mortality of severe bacterial pneumonia-associated ARDS. Therefore, new strategies focusing on host mechanisms are urgently needed and generally accepted for treating bacterial pneumonia. The immune system has evolved to allow hosts to control and eliminate pathogens.⁴ The majority strategy of host defense against pathogens involves resistance mechanisms that attack the pathogen to block invasion, kill and eliminate the offending organism.⁵ There is mounting evidence that bacterial cytotoxicity does not be directly enough to cause disease severity or mortality of the victim, but results from the manifestation of the host’s immune response, commonly referred to as cytokine storm.⁶ The antipathogen response can cause critical and even fatal tissue injury, that is, immunopathology.⁷ In contrast to resistance to infection, modification of host defense by enhancing the tolerance to bacterial invasion seems more efficient than only targeting the pathogen specifically. Thus, it is important to reveal the mechanism of controlling infection by limiting host-induced bystander damage.

Macrophages respond to pathogens and other noxious stimuli by pathogen-associated molecular patterns (PAMP) and damage-associated molecular patterns (DAMP), respectively, and initiate innate immune responses in the lung.⁸ Macrophages could induce phagocytosis, and release reactive oxygen species (ROS) and nitrogen intermediates to directly kill bacteria.⁹ On the other hand, it could produce massive proinflammatory, and anti-inflammatory cytokines, and chemokines, leading to consequent neutrophils recruitment and bacteria killing.¹⁰ The appropriate immune response is a benefit for bacteria clearance. In fact, exacerbated and persistent inflammatory responses exist in bacterial pneumonia, causing severe lung damage.¹¹ Large amounts of studies described that continuous and dysregulated macrophage activation is a vital step for aggravating the progression of pneumonia, such as COVID-19, influenza, and bacterial infection.¹²^,¹³^,¹⁴ It is interesting to note that anti-cytokine therapy, has been reported to inhibit the activated macrophage-induced production of interleukin-6 (IL-6) or IL-1 to treat pneumonia, including COVID-19.¹⁵^,¹⁶ However, few specific drugs to target macrophage activation in pneumonia have been discovered.

FcγR, binding the Fc portion of immunoglobulin G (IgG), triggers activating and inhibitory signaling pathways, which sets thresholds for cell activation and thus generates a well-balanced immune response.¹⁷ Mouse FcγRs consist of activating FcγRs (FcγRI, III, and IV) and inhibitory FcγR (FcγRIIB). The role of the FcγR family is mirrored by four different subclasses in mice, including IgG1, IgG2a, IgG2b, and IgG3.¹⁷^,¹⁸ Mouse IgG1 binds to FcγRIIB with higher affinity, while IgG2a binds to activating FcγRs, especially FcγRI, with less affinity with FcγRIIB.¹⁹ Activating FcγRs are associated with the common signaling adapter FcRγ, which contains an immunoreceptor tyrosine-based activation motif (ITAM) and is the prime effector of the proinflammatory activity of IgG in inflammation. By contrast, FcγRⅡB is a single-chain inhibitory FcγR, containing an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic region that is phosphorylated and recruits the inositol 5-phosphatase (SHIP-1 and SHIP-2) to negatively regulate innate and adaptive immunity. Several studies demonstrated that ITAM could also initiate inhibitory signaling without co-ligation with heterologous receptors, named inhibitory ITAM (ITAMi).²⁰ Notably, cell activation only ensues after the receptors have been cross-linked by antigen. In the absence of sustained aggregation, receptors show anti-inflammatory effects.²¹ Monovalent or divalent targeting of FcRs bearing an ITAM motif induced ITAMi signals that involved activation and recruitment of the Src homology region 2 domain-containing tyrosine phosphatase-1 (SHP-1), after specific targeting of ITAM-bearing receptor with a weakly binding ligand.²²^,²³

Previous studies have shown that the activation of FcγR on macrophages by immune complexes (IC)-IgG leads to cytokine signaling and inflammatory pathogenesis in rheumatic arthritis,²⁴ systemic sclerosis,²⁵ vascular inflammation, and abdominal aortic aneurysm development.²⁴^,²⁶ The signal regulated by FcγR has a profound impact on the function of monocytes and macrophages and is also involved in ARDS. For example, Menna R et al. reported that FcγRⅡB-deficient mice showed increased phagocytosis of pneumococci by macrophages in vitro and increased bacterial clearance and survival in vivo. However, previously immunized FcγRⅡB-deficient mice challenged with large inocula showed reduced survival. This correlated with increased production of the sepsis-associated cytokines tumor necrosis factor-α (TNF-α) and IL-6.²⁷ Thus, FcγRⅡB controls the balance between efficient pathogen clearance and the cytokine-mediated consequences of sepsis.²⁷ Fabiano et al. found that FcγRIII bound Escherichia coli and this interaction induced FcRγ phosphorylation, recruitment of the tyrosine phosphatase SHP-1 and phosphatidylinositide-3 kinase (PI3K) dephosphorylation. Decreased PI3K activity inhibited E. coli phagocytosis and increases TNF-α secretion of macrophages through toll-like receptor 4 (TLR4). The absence of FcγRIII protected mice from E.coli sepsis.²⁸ The recent study found that the uptake of antibody-opsonized SARS-CoV-2 by FcγRs on monocytes and macrophages triggered inflammatory cell death (pyroptosis) that aborted the production of the infectious virus but caused systemic inflammation that contributed to COVID-19 pathogenesis.²⁹ Thus, it is crucial to inhibit FcgR-mediated macrophage activation whether in autoimmune diseases or infectious-associated ARDS. It is an attractive therapeutic strategy to inhibit FcγR activation for treating autoimmunity. For example, the treatment of FcγR-blocking antibody could inhibit K/BxN arthritis and active systemic anaphylaxis.³⁰ Besides, intravenous immunoglobulin (IVIg) is widely used in clinical treatment for autoimmune and inflammatory diseases.³¹ Meanwhile, the IgG-Fc fragment has been demonstrated good efficacy in animal models of rheumatoid arthritis and human with immune thrombocytopenia.³²^,³³ However, whether inhibiting the FcγR activation of macrophages in the lung could protect against bacterial pneumonia remains unclear.

This study was to produce a therapeutic for bacterial pneumonia that specifically inhibits macrophage activation by disturbing FcγR and clarify the mechanism. In the previous study, we carried out the study of gene expression profile on mouse lung tissue of bacterial ARDS. We focused on the key host defense gene PGLYRP1 and FcγR in the process of lung injury and repair via the analysis of time series and Gene Ontology (GO) enrichment. PGLYRP1 could recognize and bind to peptidoglycan of gram-positive bacteria and the outer membrane and LPS of gram-negative bacteria and plays an important role in regulating the innate immune response.³⁴ Recent studies showed broad antibody-like reagents against microorganism infection, where the binding domains recognizing pathogens fused with the Fc portion (Hinge-CH2-CH3) of an antibody.³⁵^,³⁶^,³⁷ Here, we designed antibody-like molecules by creating IgG Fc fusions with PGLYRP1. In this study, we found that mouse IgG2a-Fc fusion with the extracellular region of PGLYRP1(PGLYRP1-Fc) binds to the macrophages and inhibited the production of proinflammatory cytokines. Besides, PGRLYRP1-Fc could improve lung injury and reduce the mortality of mice from challenges with E.coli via regulating aggravated inflammatory response and not affecting the bacterial load. Mechanistically, our results demonstrated that PGRLYRP1-Fc suppressed NF-κB activation via AKT-mediated signaling, the downstream of FcγR activation. Therefore, PGRLYRP1-Fc as a specific inhibitor of macrophage activation might be a potential intervention for the prevention and treatment of ARDS by enhancing host tolerance against bacterial infections.

Results

Construction, production, and functions of Fc-fusion proteins

Although no antibacterial IgG was produced after bacterial infection for 24 h, our previous study found that the expression of FcγR, including FcγRⅠ, FcγRⅡB, FcγRⅢ, and FcγRⅣ, the receptor of IgG, was significantly upregulated in the lung during bacterial infection. We assumed that FcγR-mediated immune response was involved in ARDS. To test the hypothesis, we designed an antibody-like molecule, PGLYRP1-Fc (Figure 1A). PGLYRP1-Fc was produced by fusing a mouse IgG2a Fc with the extracellular region of PGLYRP1. PGLYRP1 was placed at the N terminus of the Fc region (Figure 1B). Consistently, mouse IgG1 Fc was fused with PGLYRP1 to generate PGLYRP1-mIgG1-Fc. As a control, we produced mouse IgG2a-Fc (Fc only) and fused human IgG1-Fc-mut (a mutation of the amino acid residues in the Fc region) with PGLYRP1 (PGLYRP1-Fc-M). The recombinant proteins mentioned above were produced in the Expi-CHO cell expression system and purified by protein G affinity chromatography. Like typical single-chain antibodies, the recombinant proteins formed the expected dimers that were stabilized by disulfide bridges, as showed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Figure S1) and Western blot analysis (Figures 1C and 1D). Elimination of disulfide bridges with BME led to products with one-half the molecular mass (Figures 1C and 1D).

Next, we used flow cytometry to analyze the binding of purified proteins to FcγR on macrophages. The recombinant proteins were incubated with cell line Raw264.7 expressing FcγRs. The results of the flow-cytometric analysis showed that only PGLYRP1- Fc could bind to murine macrophages (Figure 1E). Besides, PGLYRP1-Fc is bound to Raw264.7 cells in a concentration-dependent manner (Figure 1F). LPS stimulated cells to flatten into a round, pancake-like shape (type 1 macrophage phenotype, M1) within 24 h, while PGLYRP1-Fc promoted cellular elongation (M2 phenotype) and changed LPS-induced morphology³⁸ (Figure S4A). What’s more, the result of real-time PCR demonstrated that PGLYRP1-Fc inhibited the transcriptional regulation of LPS-induced IL-6 and C-X-C motif ligand 2 (CXCL2) (Figure S4B). Therefore, these discoveries preliminarily suggest that PGLYRP1-Fc could function as an effective inhibitor of macrophage activation.

PGLYRP1-Fc protects against lethal acute bacterial pneumonia

To test whether PGLYRP1-Fc is protected against bacterial infection, a lethal murine ARDS model was built by injecting live bacteria. Our experiment (0.5, 1.5, and 5 mpk, −2 h, 0 days）found that PGLYRP1-Fc did not reduce the mortality of ARDS mice, and medium dose (1.5 mpk, −2 h, 0 days) could decelerate the death process of ARDS mice (Figure S5). We considered that a lethal dose of bacteria given to the lung by intratracheal injection at one time belonged to infection from severe to death (not a progressive process from mild to severe) at the time of the outbreak. The disease developed rapidly (within hours), without symptomatic treatment, such as intensive Care Unit (ICU), fluid replacement, ventilator, and cardiopulmonary resuscitation. Consequently, PGLYRP1-Fc should be injected at least 24 h in advance. Mice were then subcutaneously (s.c) injected with PGLYRP1-Fc (1 mg per kg, mpk) 3, 2, or 1 day in advance, once a day. Mice were then i.t. administered with 100 × 10⁶ colony-forming units (CFU) of live E.coli diluted in 50 μL saline at day 0 (Figure 2A). The infected mice were monitored for survival data. PGLYRP1-Fc pretreatment (1mpk, −2, -1day) significantly improved survival rates, compared with buffer control (Figure 2B). The results showed that these three ways of drug administration could reduce the mortality of ARDS mice, and there was no significant difference between the two groups.

PGLYRP1-Fc improves lung inflammation and injury without affecting the bacterial burden

Severe tissue inflammation is associated with exaggerated lung injury and mortality. A sublethal bacterial pneumonia model was used to investigate the effect of PGLYRP1-Fc on lung inflammation and injury. We used the administration scheme of Figure 2A to explore the preventive effect of PGLYRP-Fc on lung injury and inflammation in mice infected with a non-lethal dose of bacteria. We found that PGLYRP-Fc dramatically reduced the white blood cells (WBCs) and neutrophil aggregation of bronchioalveolar lavage fluid (BALF) at 24 h post-infection (hpi), but did not significantly reduce the number of red blood cells (RBCs) and the total protein of BALF (the indicators of lung injury). In addition, due to the absence of PGLYRP1-Fc treatment after bacterial infection, the number of neutrophils in the BALF of mice in the PGLYRP1-Fc group increased at 48 hpi, which was lower than the mice in the control group at 24 hpi (Figures S6A–S6D). Therefore, we adjusted the drug administration (1mpk, −1, 0 days), that is, firstly subcutaneous injection with PGLYRP1-Fc (1mpk) one day in advance (−1 day), and given PGLYRP1-Fc (1mpk) once after bacterial infection (0 days), to observe the lung injury and inflammation in ARDS mice (Figure 3A). The result of disease severity by accessing behavioral and physiological scores, in which higher scores reflected increasing disease severity,³⁹ indicated that PGLYRP1-Fc dramatically reduced the severity of the mice’s illness (Figure 3B). In addition, analysis of BALF from the total lungs revealed that bacterial instillation resulted in increased total leukocytes in both infected groups, compared with the uninfected mice at 24 hpi (Figure 3C). Wright-Giemsa staining identified that neutrophils were the main subsets of leukocytes, and PGLYRP1-Fc-treated mice presented fewer numbers of neutrophils (Figure 3D). Interestingly, PGLYRP1-mIgG1-Fc also reduced neutrophil recruitment, which might result from the binding to neutrophils, while mIgG2a-Fc and PGLYRP1-Fc-M had no significant influence on the number of neutrophils after bacteria challenge (Figures S7A and S7B). Immunohistochemistry of myeloperoxidase (MPO), released from dying neutrophils, further validated that the recruitment of neutrophils greatly decreased in PGLYRP1-Fc treated lung tissues (Figures 3K and 3L). Excessive phagocytes and apoptotic neutrophils could not be cleared in time, and newly stimulated neutrophils continue to flow into the infected lung, resulting in the harmful residence of neutrophils and extensive alveolar-capillary damage.⁴⁰ The enhanced severity of ARDS was also determined using several indices including increased levels of hemorrhage, alveolar protein accumulation, and levels of cytokines in BALF.⁴¹ PGLYRP1-Fc reduced the number of RBCs and the levels of BALF total protein (Figures 3E and 3F). Bacterial infection significantly led to increased expression of IL-6, TNF-α, and CXCL2. The results of the enzyme-linked immunosorbent assay (ELISA) indicated that PGLYRP1-Fc inhibited the production of IL-6 in BALF (Figure 3G) and lung homogenate (Figure 3M). In addition, the treatment of PGLYRP1-Fc restrained the mRNA expression of IL-6, TNF-α, CXCL2, and triggering receptor expressed on myeloid cells 1 (TREM1) in total cells of BALF (Figure 3J). Interestingly, the secretion of TNF-α and CXCL2 in BALF and lung homogenate kept at higher levels in whether PGLYRP1-Fc group or buffer control group than in the sham group (Figures 3H, 3I, 3N, and 3O). Importantly, we found that the treatment of PGLYRP1 did not adversely affect bacterial clearance (Figures 3P and 3Q). Overall, these results confirm that PGLYRP1-Fc is critical to ameliorating the severity of acute pneumococcal pneumonia mice and improving survival rates.

PGLYRP1-Fc ameliorates pathological lung damage during bacterial lung infection

To clearly evaluate the effect of PGLYRP1-Fc on tissue damage and severe pathology, histopathological examination was performed on the entire lungs of mice after different infectious times. After E.coli infection, mice were given PGLYRP1-Fc (0.3 mpk or 0.15 mpk, once a day) by subcutaneous injection at 0–3 days and 4–5 days, respectively (Figure 4A). Hematoxylin and eosin (H&E) staining of the lung at 1, 3, 5, and 7-day post-infection (dpi) indicated that there was serious edema, hyperemia, inflammatory cell recruitment, and destruction of alveolar structure in the infectious lung within 5 days after bacterial exposure (Figures 4B–4G). Untreated-lung pathology began to recover after 7 dpi. The treatment of PGLYRP1-Fc after bacterial infection could markedly ameliorate pathological features and effectively resume alveolar structure (Figures 4C–4G). This provides additional support for the role of PGLYRP1-Fc in protecting mice from lung injury induced by bacterial challenge.

PGLYRP1-Fc regulates LPS-induced inflammatory response in macrophages and inhibits activated-macrophages induced-neutrophil migration

Macrophages play a critical role in initiating and resolving inflammation during bacterial pneumonia.⁹ Disturbing macrophage activation is becoming a potential way to treat acute lung injury/ARDS.¹¹ We further investigated whether PGLYRP1-Fc could directly affect macrophage activation. Macrophage activation is under the control of signaling cascades that are initiated by toll-like receptors (TLR) and cytokine signals, resulting in the production of pro- and anti-inflammatory mediators.⁸ Given that PGLYRP1-Fc could bind E.coli and LPS in a concentration (0.01–3 μM/mL)-dependent manner and the binding at 1 μM/mL was higher (Figures S2A and S2C). Besides, PGLYRP1-Fc at a concentration lower than 40 μM/mL did not affect the vitality of macrophages (Figure S3). Consequently, we chose to float up and down at 1 μM/mL to experiment in vitro. PGLYRP1-Fc alone was not sufficient to alter the levels of proinflammatory and anti-inflammatory cytokines, while it significantly decreased the transcripts of proinflammatory cytokines including IL-6, TNF-α, and chemokines, such as CXCL1, CXCL2, and C-C motif chemokine ligand 2 (CCL2), which were upregulated by LPS (Figures 5B, 5C, and 5G–5I). Besides, secreted inflammatory cytokines including IL-6, TNF-α, and CXCL2 were all significantly decreased in PGLYRP1-Fc-treated macrophages (Figures 5E–5F and 5J). Interestingly, PGLYRP1-Fc further elevated the production of LPS-induced anti-inflammatory IL-10 in primary macrophages (Figures 5A and 5D). The secretion of IL-10 in the untreated and PGLYRP1-Fc-treated cells is lower than the detection limit, not shown. In addition to regulating inflammatory cytokine expression, PGLYRP1-Fc significantly depressed the expression of LPS-stimulated inducible nitric oxide synthase (iNOS) (Figure 5K), a key enzyme in catalyzing the production of nitric oxide (NO), contributing to inflammation and the killing of pathogens.⁴² TREM1 potently amplifies proinflammatory cytokine secretion.⁴³ LPS treatment induced-overexpression of TREM1 was dramatically down-regulated by PGLYRP1-Fc (Figure 5L).

PGLYRP1- Fc regulates the expression of LPS-induced inflammatory response by macrophages and inhibits macrophage-induced neutrophil migration after LPS stimulus

The peritoneal macrophages were treated with PGLYRP1-Fc or/and LPS at the indicated concentrations for 24 h.

(A–C), (G–J), (K), and (L) Real-time PCR analyses of inflammatory mediators, including IL-6, IL-10, TNF-α, CXCL1, CXCL2, CCL2, iNOS, and TREM1.

(D–F) ELISA analysis of the secretion of IL-6, IL-10, and TNF-α in cell supernatant.

(M) The ability of macrophages to recruit neutrophils was determined by the number of cells of the lower well under a microscope (original magnification 100×). Scale bar, 100 μm.

(N) The effect of PGLYRP1-Fc on the neutrophil migration rate in the co-cultured neutrophils and cell supernatant of macrophages Transwell system. The migration of neutrophils was counted using a hemocytometer. Data are expressed as mean ± SEM (n = 3), ∗∗p < 0.01 buffer control, and ∗∗∗p < 0.001 buffer control. #p < 0.05 versus LPS, ##p < 0.01 versus LPS, and ###p < 0.001 versus LPS. See also Figures S2, S3, and S8–S10.

The effect of macrophage on neutrophil migration in vitro was determined by transwell assay. The primary macrophages cultured in serum-free RPMI-1640 medium were pretreated with PGLYRP1-Fc or/and LPS for 24 h. The medium was collected and centrifugated. Transwell migration assay was performed in a co-culture system of neutrophils and cell supernatant of macrophages. As shown in Figures 5M and 5N, LPS-stimulated cell supernatant of macrophages markedly promoted neutrophil migration compared with negative control, and PGLYRP1-Fc treatment significantly impaired this effect. Collectively, these results suggest that PGLYRP1-Fc has the potential to inhibit macrophage activation and suppress macrophage-induced neutrophil migration by blocking proinflammatory signals.

PGLYRP1-Fc suppresses inflammatory response in macrophages via AKT mediated NF-κB pathway

To further clarify the mechanistic signaling that underly the inhibition of macrophage activation, we examined the activation of transcription factors NF-κB and AKT in regulating inflammatory cytokines. Figure 6A showed that the phosphorylation of NF-κB-p65 and AKT decreased after PGLYRP1-Fc treatment, and the down-regulated phosphorylation level of NF-κB-p65 and AKT was sustained for 24 h. Besides, PGLYRP1-Fc significantly reduced the phosphorylated expression of p65 and AKT in dose-dependent manners (Figure 6B). In addition, PGLYRP1-Fc impaired the effect of LPS on upregulating the phosphorylation of NF-κB-p65 and AKT in vitro and ex vivo (Figures 6C and 6D). The nuclear translocation of NF-κB-p65 is a key step in regulating inflammatory response.⁴⁴ Immunofluorescence staining of NF-κB determined that PGLYRP1-Fc inhibited LPS-induced nuclear translocation of p65 in macrophages (Figure 6E). Next, we explored the role of AKT signaling in controlling the activity of NF-κB. Selective AKT agonist SC79 markedly increased the protein expression of phosphorylated NF-κB-p65 and the nuclear translocation of p65 (Figures 6F and 6G), while PGLYRP1-Fc treatment dramatically restrained the activation of NF-κB (Figures 6F and 6G). Taken together, these findings show that PGLYRP1-Fc inhibits macrophage activation via suppressing AKT-regulated NF-κB signaling.

PGLYRP1-Fc suppresses inflammatory response in macrophages via the AKT-NF-κB pathway

(A) The peritoneal macrophages of WT mice were treated PGLYRP1-Fc at 1 μM for different hours or the indicated doses for 24 h. Western blot analyses of phosphorylated NF-ΚB-p65 (Ser526), AKT (Ser473), and corresponding total protein expression in the macrophages. The peritoneal macrophages of WT mice were treated with PGLYRP1-Fc or/and LPS at the indicated concentrations for 24 h.

(B and C) Immunoblot analysis of phosphorylated-AKT, AKT, phosphorylated-NF-κB-p65, and NF-κB-p65 was performed.

(E) The nuclear translocation of NF-κB-p65 was assessed by immunofluorescence. Scale bar, 50 μm.

(D) C57BL/6N mice subcutaneously received buffer or PGLYRP1-Fc (1 mpk) daily for 2 days and then the peritoneal macrophages were isolated and stimulated by LPS (100 ng/mL) for 24 h. Western blot analysis of phosphorylated-AKT, AKT, phosphorylated-NF-κB-p65, and NF-κB-p65. The peritoneal macrophages of WT mice were treated with PGLYRP1-Fc or AKT agonist SC79 at the indicated concentrations for 3 h.

(F) Phosphorylated-AKT, AKT, phosphorylated-NF-κB-p65, and NF-κB-p65 were analyzed by Western blot.

(G) Immunofluorescence staining for NF-κB-p65. Scale bar, 50 μm. Data are expressed as mean ± SEM (n = 3), ∗p < 0.05 versus buffer control, ∗∗p < 0.01 versus buffer control, and ∗∗∗p < 0.001 versus buffer control. #p < 0.05 versus LPS, and ##p < 0.01 versus LPS.

PGLYRP1-Fc reduces lung inflammation via AKT/NF-κB in mice

To understand the role of PGLYRP1-Fc in controlling lung inflammation, studies were performed to determine whether PGLYRP1-Fc regulates AKT/NF-κB signaling pathway in vivo. Results from dual immunofluorescence staining of murine lung tissues displayed that PGLYRP1-Fc decreased the expression of phosphorylated NF-κB-p65 induced by E.coli infection (Figure 7A). Besides, this staining indicated a close co-localization of phosphorylated-p65 with macrophage maker F4/80⁴⁵ (Figure 7A). We also examined the protein abundances of AKT and NF-κB-p65 in mice lung tissues. Bacterial-infected mice presented increased total protein expression of p65 and higher phosphorylation levels of AKT but significantly reduced in PGLYRP1-Fc-treated mice when infected with E.coli (Figures 7). Therefore, our data confirm that PGLYRP1-Fc attenuates lung inflammation dependent on AKT/NF-κB signaling in macrophages.

PGLYRP1-Fc ameliorates inflammatory response via inhibiting the NF-κB pathway in mice

Mice were first subcutaneously injected with PGLYRP1-Fc (1 mpk) or buffer control one day in advance, and i.t. administered with 50 × 10⁶ CFU of *E.coli* diluted in 50 μL saline or 50 μL saline and then given PGLYRP1-Fc (1 mpk) or buffer control by subcutaneous injection at day 0. The mice’s lung was collected at 1 dpi.

(A) Immunofluorescence staining of the left lung sections using antibodies against phosphorylated-NF-κB-p65. Scale bar, 50 μm.

(B) Western blot analysis of phosphorylated-AKT, AKT, phosphorylated-NF-κB-p65, and NF-κB-p65 expression from the right lungs of WT and PGLYRP1-Fc treated mice with or without bacterial challenge at 1 dpi.

(C and D) Phosphorylated-AKT, phosphorylated-NF-κB-p65 and corresponding total protein expression was quantified by Image J. Data are expressed as mean ± SEM (n = 3), ∗p < 0.05 versus sham, and ∗∗p < 0.01 versus sham. #p < 0.05 versus *E.coli*,and ##p < 0.01 versus *E.coli*.

PGLYRP1-Fc impairs the bactericide and phagocytosis of macrophages and does not affect the bactericidal ability of neutrophils

Given that PGLYRP1-Fc did not affect the bacterial burden in BALF and lung tissues, we further assessed the bacterial killing of different immune cells. Peritoneal macrophages from wild-type (WT) mice were pretreated with PGLYRP1-Fc or/and LPS for 24 h and then incubated with luminescent-E.coli in vitro, and bacterial titers were detected by luminescence. PGLYRP1-Fc dose-dependently inhibited the bacterial killing response of macrophages (Figures 8A and 8B), while PGLYRP1-Fc-M did not alter the ability of bacterial killing of macrophages. Besides, PGLYRP1-Fc and PGLYRP1-Fc-M did not alter the ability of bacterial killing in peripheral blood cells, where granulocytes accounted for the main composition (Figures 8D and 8E). Consistently, ex vivo assay for bacterial killing showed that PGLYRP1-Fc treated mice had an impaired ability to kill bacteria by alveolar macrophage, compared with WT mice (Figure 8C). What’s more, total BALF cells from mice i.t with E.coli (10 × 10⁶ CFU/mice) 3 days in advance, which consist of alveolar macrophages and neutrophils, were harvested from infected mice. The BALF cells from infectious mice had enhanced bactericidal ability, compared with unstimulated BALF, which showed that neutrophils had a strong bactericidal ability. However, PGLYRP1-Fc did not affect the bactericide of BALF cells from E.coli-challenged mice, consistent with the result in vivo (Figure 8C). In addition, preincubation of the primary macrophages by PGLYRP1-Fc not only inhibited the phagocytosis of E.coli in a concentration-dependent manner but also significantly reduced the phagocytosis of E.coli induced by LPS stimulation (Figures 8F and S11).

Discussion

Lung bacterial infection associated with ARDS leads to significant morbidity and mortality, especially in the elderly. Although antibiotics provide pharmacologic-driven resistance as a means against pathogens, multidrug-resistant strains still escape antimicrobial drugs. Besides, pathogens could escape the hosts’ immune resistance, and immune intervention aiming to reduce the pathogen load induces stress and damage to the host tissue, which increases the morbidity and mortality of ARDS. In contrast to the process of immune resistance, host tolerance renders immune cells unresponsive and dampens tissue damage without affecting pathogen clearance. We argued that the strategy for enhancing host tolerance might help to limit infectious disease severity. In this study, we designed an antibody-like molecule by creating mouse IgG2a Fc fusions with PGLYRP1. We confirmed that PGLYRP1-Fc protected against lethal acute bacterial pneumonia and improved lung inflammation and injury, without affecting the bacterial load. Specifically, we discovered that PGLYRP1-Fc bound to macrophages, depending on the Fc portion. Besides, PGLYRP1-Fc exerted an inhibition on the production and secretion of proinflammatory cytokine in LPS-stimulated-macrophages, and restrained neutrophil migration in vitro. We also demonstrated that the suppressive effect of PGLYRP1-Fc on controlling inflammatory response in LPS-induced macrophage during bacterial pulmonary infection is mediated by the AKT/NF-κB axis, the downstream of the FcγR.

Recent studies showed broad antibody-like reagents against microorganism infections, where the binding domains recognizing pathogens fused with the Fc portion (Hinge-CH2-CH3) of an antibody.³⁵^,³⁶^,³⁷ However, these antibody-like molecules have high specificity and usually target limited antigen epitopes, which limits the recognition of other microorganisms. PGLYRP1 can recognize the peptidoglycan of gram-positive bacteria and the outer membrane and LPS of gram-negative bacteria. By optimizing the antigen binding domain, we designed an IgG-like molecule PGLYRP1-Fc, in which PGLYRP1 bound bacteria and Fc bound FcγRs. FcγRs on immune cells recognize the Fc region of IgG and induce a widely downstream effector, such as phagocytosis, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), superoxide production, as well as inflammation inhibition, all of which depend on the ligand and cell types.¹⁷ PGLYRP1-Fc might protect against multiple bacterial infections. Mouse high-affinity FcγRⅠ could bind monomeric IgG, both high-affinity FcγRⅠ and the medium/low-affinity FcγRⅡB, FcγRⅢ, and FcγRⅣ bind multivalent IgG with high avidity. The presence of the Fc region is also used for the purification through immunoaffinity chromatography based on protein G. We found that the purified fusion proteins are expressed as a dimer in solution and PGLYRP1-Fc bound to E.coli and LPS dose-dependently within a certain concentration range (Figures S2A and S2C). However, PGLYRP1-Fc did not promote the opsonophagocytic killing of E.coli (Figure S2B). In line with Gillian Dekkers et al.’s report, they showed the binding of mIgG1 to FcγRⅡB and FcγRⅢ, which is equally well, and that mIgG2a bound all receptors with FcγRⅠ＞FcγRⅣ＞FcγRⅢ＞FcγRⅡB,¹⁹ we identified that PGLYRP1-Fc bound to macrophages (express FcγRI-IV) and PGLYRP1-mIgG1-Fc bound to neutrophils (express FcγRⅡB and FcγRⅢ). Cell morphology also showed that only PGLYRP1-Fc promoted cellular elongation (M2 macrophage) and changed LPS-induced pancake-like shape (M1 macrophage). In addition, PGLYRP1-Fc inhibited LPS induced-IL-6 expression specifically, while PGLYRP1-Fc-M further upregulated the expression of IL-6, IL-10, TNF-α, and TREM1 in LPS-treated macrophages (Figures S4B and S10A–S10C), which was consistent with the previous studies that PGLYRP1 could function a ligand as TREM1, a receptor expressed on macrophages, and multivalent stimulation of TREM1 initiated innate immunity, after priming by LPS.⁴⁶^,⁴⁷ Mouse IgG2a-Fc and PGLYRP1-mIgG1-Fc have no influence on macrophages after LPS stimulus in the indicated concentrations, which might be related to the lower affinity of monovalent IgG2a and IgG1 binding to the FcγRs on macrophages. These data suggested that PGLYRP1-Fc inhibited proinflammatory response in LPS-stimulated macrophages specifically. The current antibody-like molecules protected infectious mice by neutralizing and killing specific pathogens and the resistance of viruses or bacteria to existing antibodies due to their high mutation rates increased. The modification of host immune response by PGLYRP1-Fc to multiple pathogen invasion seems more promising than that of directly targeting the pathogens. Next, we focused on exploring the effect of PGLYRP1-Fc on bacterial pneumonia and macrophage function.

Accumulating evidence reported that during bacterial infection, excessive macrophage activation and subsequently increased infiltration of neutrophils triggered the production of extensive cytokines, favoring bacteria growth, in which bacteria might adapt to the microenvironment by utilizing cytokines as their growth factors.⁴⁸ On the other hand, massive inflammation could damage the tissue, impairing the ability of the host to remove bacteria. In fact, the lower levels of proinflammatory cytokines were efficient to kill bacterial.⁴⁸ Herein, a lethal murine bacterial pneumonia model was built. We found that the pretreatment of PGRPLY1-Fc significantly reduced the mortality in mice after the E.coli challenge. Besides, PGRPLY1-Fc intervention ameliorated bacterial infection-induced exaggerated lung hemorrhage, injury, and inflammation. However, the bacterial load changed neither in BALF nor lung homogenate in PGLYRP1-Fc treated mice after E.coli infection, compared with pneumonia mice. Our results in vitro and ex vivo revealed that PGLYRP1-Fc impaired the bactericidal ability of macrophages, associated with impaired phagocytosis, and did not affect the bacterial killing of neutrophils. Neutrophils played a key role in killing bacteria and macrophages mainly functioned as regulating inflammatory response during bacterial infection. Although PGLYRP1-Fc reduced neutrophil recruitment to the infectious site, the remaining neutrophils were sufficient to control bacteria, and the decrease in neutrophil recruitment was also beneficial to improve lung damage. Importantly, the histopathological analysis indicated that PGRPLY1-Fc treatment effectively improved the pathological characteristics of pneumonia and resumed alveolar structure, even when used after the onset of infection. In line with previous reports, the researchers found that epirubicin had a protective effect in septic mice via strengthening disease tolerance, related to cytokine inhibition.⁴⁹ Our results showed that PGLYRP1-Fc protected against bacterial pneumonia by promoting host tolerance with reduced inflammatory response and tissue damage, irrespective of the host-pathogen burden, which provided a new candidate drug for bacterial infection.

Given that PGLYRP1-Fc bound to macrophages dependent on the Fc region, we assumed that PGLYRP1-Fc suppressed LPS-induced inflammatory response via the FcγR-mediated signaling cascade. Firstly, we confirmed the effect of PGLYRP1-Fc on macrophage activation. PGLYRP1-Fc alone was not sufficient to alter the levels of proinflammatory mediators, including IL-6, TNF-α, IL-10, and chemokines, but significantly downregulated LPS-stimulated transcription and secretion of IL-6, TNF-α, and chemokines and further promoted the expression of anti-inflammatory cytokine IL-10 in primary macrophages. The change of proinflammatory cytokines is usually followed by the consistent change of anti-inflammatory cytokines as a feedback loop.⁵⁰ PGLYRP1-Fc further increased LPS-induced IL-10 expression in primary macrophages, aiming to maintain homeostasis of inflammatory response. PGLYRP1-Fc skewed LPS-induced type 1 macrophage phenotype toward M2 macrophage, as decreased expression of iNOS, further increased expression of IL-10 and CD206⁵¹ (Figure S8), which was consistent with the cell morphology. The results in the Raw264.7 cell line were consistent with the above (Figures S9A and S9C–S9F). These data further reveal that ‘unresponsive’ lung pretreated with PGLYRP1 presents reduced inflammation responding to bacteria. Notably, LPS-induced IL-10 expression was further inhibited by PGLYRP1-Fc in Raw264.7 (Figure S9B). We speculated that the inflammation level is different between Raw264.7 cells and primary macrophages after the LPS stimulus. The down-regulated expression of proinflammatory cytokines such as IL-6 by PGLYRP1-Fc was efficient to restore homeostasis in Raw264.7 cells, followed by the consistent change of IL-10 as a feedback loop.⁵⁰ Christine B et al. reported that multimerized PGLYRP1 was identified as a functional TREM1 ligand.⁴⁶ It is well-known that TREM1 is a proinflammatory receptor expressed on macrophages/monocytes and neutrophils, and its expression is upregulated during bacterial infection. TREM1 could synergize with TLR ligands to amplify inflammatory responses.⁴⁷ Consistently, soluble PGLYRP1-Fc or PGLYRP1-Fc-M did not induce a stimulant of TREM1. Interestingly, PGLYRP1-Fc inhibited LPS-induced TREM1 activation, which was further upregulated by PGLYRP1-Fc-M, not only in primary macrophages (Figure S10B) but also in the Raw264.7 cell line (Figure S9E). This result also suggested that PGLYRP1-Fc regulated inflammatory response in macrophages mainly dependent on the interaction with FcγR. The primary mechanism of neutrophil chemotaxis to infection sites is the recruitment of activated-macrophage, especially in the alveoli where macrophages account for almost 100% of cells in BALF.⁵² Consistent with the result in vivo, proinflammatory cytokines produced by activated-macrophage promoted neutrophil migration in vitro, and PGLYRP1-Fc inhibited macrophage-induced neutrophil migration after LPS treatment.

Usually, activated FcγRs bound IgG-IC and induced proinflammatory signaling, mediated by ITAM, while FcγRⅡB containing ITIM negatively regulates innate and adaptive immunity. In the absence of sustained aggregation, receptors show anti-inflammatory effect. Recent studies showed that monomeric IgG1 interacted with low-affinity FcγRⅢ, inducing ITAMi inhibition signaling, independently of antigen.²² In addition, The Fc trimer, Fc3Y did not induce cellular activation but instead inhibited FcgR-mediated responses to ICs in a wide variety of human immune cells.⁵³ Therefore, it is possible that monomeric PGLYRP1-Fc binds to high-affinity FcγRⅠ and also might interact with low-affinity FcγRIII, which induces ITAMi inhibition signaling and anti-proinflammatory response in this study. We will further investigate the mechanism by which PGLYRP1-Fc interacts with FcγR on macrophages in the next research. In the current study, we found that PGLYRP1-Fc inhibited the FcγR-mediated phosphorylation of the AKT/NF-κB pathway both in normal and LPS-stimulated macrophages, which played a critical role in cytokine production. NF-κB could translocate to the nucleus upon LPS stimulus, and subsequently activates the immune response, while PGLYRP1-Fc suppressed the nuclear translocation of NF-κB-p65 in LPS-treated macrophages. We also identified that AKT activation was a positive correlation with NF-κB translation, and PGLYRP1-Fc regulated NF-κB signaling via AKT. At the same time, the experiment in vivo demonstrated that the phosphorylation of AKT and the total protein expression of NF-κB-p65 was downregulated by pretreatment of PGLYRP1-Fc, and phosphorylated-p65 was co-localized with F4/80, the maker of macrophages in the murine lung. These findings indicate that the pretreatment of PGLYRP1-Fc inhibits AKT/NF-κB activation via the FcγR-dependent mechanism, making macrophage unresponsive, and immediately reduces proinflammatory response upon bacteria or LPS stimulus in turn. Compare with other antibody-like molecules protecting infectious mice by neutralizing and killing specific pathogens, PGLYRP1-Fc specifically inhibits macrophage activation and protects against bacterial infection by enhancing host tolerance via regulating immune homeostasis and reducing lung damage. With the increase in the resistance of viruses or bacteria to existing antibodies due to their high mutation rates, modification of host immune response by PGLYRP1-Fc to multiple pathogen invasions seems more promising than that of directly targeting the pathogens.

In conclusion, our data demonstrate that PGLYRP1-Fc could significantly ameliorate lung inflammation and damage, and improve survival rates in ARDS mice. Mechanistically, PGLYRP1-Fc inhibits inflammatory response in macrophages by blocking FcγR-dependent AKT/NF-κB axis, followed by decreased neutrophil recruitment, which ultimately enhances host tolerance and reduces lung damage, returning to immune homeostasis. The strengthened host tolerance by PGLYRP1-Fc may be a promising therapeutic strategy for Fc-engineering drug candidates for protecting against bacterial infection. These findings provide PGLYRP1-Fc as a drug candidate with a new structure and mechanism to treat ARDS.

Limitations of the study

Our study demonstrates that PGLYRP1-Fc protects against gram-negative bacteria-infected ARDS mice and whether it can ameliorate gram-positive bacteria-associated ARDS remains to be investigated. Besides, the pharmacokinetics and pharmacodynamics to stress the efficacy of this molecule require further research. In addition, the interaction between PGLYRP1-Fc and FcγR to regulate its downstream AKT/NF-κB mediated inflammatory response in the macrophages needs further exploration.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Goat Anti-Mouse IgG (H&L) - Alexa Fluor 488	Abmart, Shanghai, China	Cat# M213208
Anti-β-actin	Abmart, Shanghai, China	Cat# P30002
Goat Anti-Mouse IgG HRP	Abmart, Shanghai, China	Cat# M21001
Goat Anti Rabbit IgG HRP	Abmart, Shanghai, China	Cat# M21002
Anti-AKT	Cell Signaling Technology	Cat# 4685
Anti-p65	Cell Signaling Technology	Cat# 8242
Anti- phosphorylated-AKT	Cell Signaling Technology	Cat# 4060
Anti- phosphorylated-p65	Cell Signaling Technology	Cat# 3033
FITC-labeled Goat Anti-Rabbit IgG (H+L)	Jackson ImmunoResearch Inc, West Grove, PA, USA	Cat# 111-095-003; RRID:AB_2337972

Bacterial and virus strains

Escherichia coli	ATCC	ATCC 25922

Chemicals, peptides, and recombinant proteins

AKT agonist (SC79)	Sigma-Aldrich	Cat# SML0749-5MG
Brewer thioglycollate medium modified	Hopebio, Qingdao, China	Cat# HB5190
Bovine serum albumin (BSA)	Sigma-Aldrich, Missouri, USA	Cat# V900933-100G
Lipopolysaccharides from Escherichia coli O111:B4	Sigma-Aldrich, Missouri, USA	Cat# L4391-1MG
Trizol	Sigma-Aldrich, Missouri, USA	Cat# T9424-100ML
RPMI 1640 medium	Thermo Fisher Scientific, MA, USA	Cat# 11875119
Phosphate-buffered saline (PBS)	Thermo Fisher Scientific, MA, USA	Cat# 10010023
Fetal Bovine Serum (FBS)	Thermo Fisher Scientific, MA, USA	Cat# 10100147C
ExpiCHO-S™ expression system kit	Thermo Fisher Scientific, MA, USA	Cat# A29133
4′,6-Diamidino-2-phenylindole (DAPI)	Beyotime, Shanghai, China	Cat# C1002
Protein G agarose	Sigma-Aldrich, Missouri, USA	Cat# 11719416001
PGLYRP1-Fc	This paper	N/A

Critical commercial assays

BCA protein assay kit	Beyotime, Shanghai, China	Cat# P0012
Cell counting kit-8 (CCK8)	Beyotime, Shanghai, China	Cat# C0038
Bradford protein assay kit	Sangon Biotech, Shanghai, China	Cat# C503041
Endotoxin Assay Kit	Xia men Bioendo technology, Xiamen, China	Cat# RCR0428
Endotoxin-free plasmid extraction kit	Omega Bio-Tek Inc, USA	Cat# D6926-03
HiScript II Q RT SuperMix kit	Vazyme, Nanjing, China	Cat# R223-01
AceQ Universal SYBR qPCR Master Mix	Vazyme, Nanjing, China	Cat# Q511-02
Mouse CXCL2 ELISA Kit	MultiSciences Biotech, Hangzhou, China	Cat# EK2142
Mouse IL-6 ELISA Kit	MultiSciences Biotech, Hangzhou, China	Cat# EK206
Mouse IL-10 ELISA Kit	MultiSciences Biotech, Hangzhou, China	Cat# EK210
Mouse TNF-α ELISA Kit	MultiSciences Biotech, Hangzhou, China	Cat# EK282

Experimental models: Cell lines

Mouse: RAW 264.7 Cell line	ATCC	TIB-71
Mouse: The primary macrophages	This paper	N/A

Experimental models: Organisms/strains

Mouse: C57BL/6 N	Vital River Laboratory Animal Technology, Beijing, China	N/A

Oligonucleotides

qPCR Primers (See table below)	This paper	N/A

Recombinant DNA

pET24a-GFP	This paper	N/A
pUC18-Luciferase	This paper	N/A
pcDNA3.4-mIgG2a-Fc	This paper	N/A
pcDNA3.4-PGLYRP1-mIgG2a-Fc	This paper	N/A
pcDNA3.4-PGLYRP1-mIgG1-Fc	This paper	N/A
pcDNA3.4-PGLYRP1-hIgG1-Fc-M	This paper	N/A

Software and algorithms

GraphPad Prism	GraphPad Software, San Diego, USA	https://www.graphpad.com/
Image J	Schneider et al.⁵⁴	https://imagej.nih.gov/ij/
Figdraw	N/A	https://www.figdraw.com/

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Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact Jingjing Li (lijj@sjtu.edu.cn).

Materials availability

This study developed a novel molecule, PGLYRP1-Fc.

Experimental models and subject details

Animals

Male C57BJ/6N mice (6–8 weeks old) weighing about 20-22g were purchased from Vital River Laboratory Animal Technology (Beijing, China). The animals were housed in a temperature and humidity-controlled room with a 12-day/night cycle and free access to food and water. All experimental procedures received the approval of the Shanghai Jiao Tong University Animal Ethics Committee.

Cell culture

The primary macrophages and Raw264.7 cell line were cultured in RPMI-1640 medium with 10% Fetal Bovine Serum (FBS) with or without 1% Penicillin-Streptomycin (P/S) and incubated at 37 °C 5% CO₂.

Bacteria culture

E.coli (ATCC 25922) was stored at −80°C in Luria-Bertani (LB) containing 25% glycerol. After overnight incubation on LB agar plates, the freshly grown colony was suspended in LB overnight incubation and transferred to fresh medium at a ratio of 1:100, and then incubated for 2 h 30 min at 37°C to logarithmic growth, pelleted, and finally resuspended in sterile PBS. OD₆₀₀ was measured by Ultraviolet spectrophotometer and bacterial concentrations were assessed by serial dilutions. The standard curve was made according to OD₆₀₀ and bacterial concentration.

Method details

Construction, purification, and characterization of Fc fusion proteins

The extracellular domain sequence of PGLYRP1 was fused with the N-terminal of mIgG1-Fc, mIgG2a-Fc, and the N-terminal of hIgG1-Fc mutant fragment to construct the fusion protein expression frame. To evaluate the contribution of Fc effector functions, we introduced loss-of-function LALA-PG (L234A, L235A, and P329G) mutations into the Fc region of the human IgG1 heavy chain.⁵⁴ According to the Chinese hamster ovary (CHO) cell codon preference and mRNA hairpin structure prediction, the fusion gene sequence was optimized for codon and mRNA secondary structure. After confirming that the optimized sequence is correct, the gene was synthesized and cloned into the pcDNA3.4 expression vector.

The plasmids including pcDNA3.4-mIgG2a-Fc, pcDNA3.4-PGLYRP1-mIgG2a-Fc, pcDNA3.4-PGLYRP1-mIgG1-Fc, and pcDNA3.4-PGLYRP1-hIgG1-Fc-M were extracted according to OMEGA endotoxin-free plasmid extraction kit and transfected into CHO cells, using OptiPRO™ and ExpiFectamine™ CHO Reagent. The Fc fusion proteins were expressed by CHO cells and purified from serum-free CHO cell supernatant by affinity and size exclusion chromatography, according to the protocols from the manufacturer. Bradford protein assay kit was used to measure protein concentrations. Endotoxin level was determined using Endotoxin Assay Kit. Purified proteins were run on 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) with or without the reducing agent β-mercaptoethanol (BME) and analyzed by Western blot using anti-mouse PGLYRP1 antibody and anti-mouse IgG.

Bacteria binding assay

E.coli (ATCC 25922, 5 × 10⁸ CFU) in logarithmic growth was fixed by 2.6% polyformaldehyde, 0.012% glutaraldehyde, and 30 mM phosphate buffer pH 7.4 for 15 min at room temperature and then on the rice for 30 min. The bacteria were washed with cold PBS three times and blocked with PBS solution containing 2% BSA+1% gelatin at room temperature for 30 minutes. The bacteria were given corresponding reagents for 1 h at room temperature. Next, cells were incubated with FITC-labeled Goat Anti-Rabbit IgG (1:200 dilution, Jackson ImmunoResearch Inc, West Grove, PA, USA) for 30 min. Finally, OD₆₀₀ and fluorescence intensity were detected by Microplate Reader. The data was presented as fluorescence intensity of FITC-labeled rabbit anti-mouse IgG-Fc antibody/bacterial density (OD₆₀₀).

LPS binding assay

96 microtiter plates (Costar) were used to test the binding activity of PGLYRP1-Fc to LPS. In brief, the plate wells were incubated with a total of 100 μL of LPS (20 μg/mL) at 37°C overnight until the plate came to desiccation. Wells served as the blank control and were incubated with 100 μL of distilled water. After being blocked with 200 μL of BSA (2 mg/mL) for 2 h and washed four times with TBST (0.05% Tween-20 in TBS), the wells were incubated with serially diluted PGLYRP1-Fc and mIgG2a-Fc (negative control) (0.001–10 μM in TBS containing 0.1 mg/mL BSA) at 37°C for 3 h and then washed four times with TBST. Each well was then incubated with 100 μL of Horseradish peroxidase-conjugated Goat Anti-Mouse IgG antibody (1:5000 dilution in TBS with 1 mg/mL BSA) at 37°C for 1 h, and then washed four times with TBST. The color reaction was developed with 100 μL of 0.01% 3,3′,5,5′-tetramethylbenzidine and stopped with 50 μL of 2 M H₂SO₄. The absorbance was recorded at 450 nm by a microtiter plate reader (Tecan, Switzerland).

Cell culture and treatment

Six-eight-week-old male C57BJ/6N mice were intraperitoneally (i.p) injected with 1 mL of 3% Brewer thioglycollate medium modified (Haibo, Qingdao, China). Mice were euthanized after 4 d, and the peritoneal cavity was washed with cold DPBS without calcium and magnesium to obtain macrophages. The primary macrophages were added to a 6-well plate or 24-well plate or 96-well plate and incubated for 1 h in a 5% CO₂ and 95% air-humidified atmosphere at 37 °C. The wells were washed three times with DPBS to remove nonadherent cells. The primary macrophages and Raw264.7 cell line were cultured in RPMI-1640 medium with 10% FBS with or without 1% Penicillin-Streptomycin (P/S) and incubated at 37 °C 5% CO₂.

The opsonophagocytic killing of E.coli

The peritoneal macrophages were treated with PGLYRP1-Fc at the indicated concentrations and incubated with luminescent-E.coli at 37 °C for 30 min with shaking, and then normally incubated for 150 min. Bacterial titers were detected by luminescence.

Macrophage phagocytosis assay

E.coli cells expressing the GFP plasmid from the separate colony were scraped off the plate, and E.coli cultures were diluted 1:100 from an overnight culture and grown to an OD₆₀₀ of 0.5. Cells were harvested, washed in DPBS, and resuspended in DPBS to an OD₆₀₀ of 0.3 (∼10⁸ cells/mL). The primary macrophages were seeded at 10⁵ cells per well of a 24-well plate. After adhering to the wall for 1h, the non-adherent cells were removed, and treated with PGLYRP1-Fc or/and LPS for 24 h. The wells were washed three times with cold PBS to remove extracellular bacteria and added with the antibiotic-free medium. 100 μL bacteria (roughly 1 × 10⁷ cells) was added to the plate, and the plate was incubated at 37 °C 5% CO₂ for 30 min. The wells were washed three times with cold PBS to stop phagocytosis. Fix the cells with 4% paraformaldehyde for 10 min at room temperature. The cells were further stained with Dil and DAPI, and analyzed by microscopy to verify the presence of fluorescent E.coli within macrophages.

Bacteria infection-induced ARDS model

E.coli was cultured to the logarithmic growth period and resuspended with saline to 1000 × 10⁶ CFU/mL. Each mouse was intratracheal (i.t.) administration with 50 × 10⁶ CFU of E.coli diluted in 50 μL saline to establish a sublethal bacterial pneumonia model, while animals were challenged with 100 × 10⁶ CFU of live E.coli to assess the mortality. Sublethal infected mice were anesthetized with an injection of pentobarbital sodium (50 mg/kg) and collected total lung BALF as described previously,⁵⁵ and then sacrificed. The entire lungs were harvested in a 2 mL EP tube with 1 mL PBS.

Bacterial burden

The BALF of the entire lungs was diluted 10-fold in sterile PBS, and 10 μL of each dilution was spotted on LB agar plates. The entire lungs were mechanically homogenized and then diluted with PBS for bacterial counts. Plates were incubated at 37°C for 16 h and the number of colonies was counted and expressed as Log10 CFU per milliliter.

BALF cell count and differentiation

Total nucleated cell numbers and RBCs were determined by a hemocytometer. Cells were stained with Wright-Giemsa for cell differentiation.

Lung histopathology and immunohistochemistry

After trachea ligation, the entire lungs were quickly removed, fixed with 4% paraformaldehyde (PFA), and subsequently embedded with paraffin for H&E staining as the previously established method.⁵⁶ Lung injury was assessed by the scoring system suggested by the research.⁵⁷ The severity of lung injury was quantified based on the findings in twenty randomly selected high-power fields (400×) for each section by a pathologist, who was blinded to the experimental protocol. Lung injury was graded from 0 (normal) to 2 (severe) for the following per field: (A) alveolar neutrophils, (B) interstitial neutrophils, (C) hyaline membrane formation, (D) presence of proteinaceous debris in the alveolar space and (E) thickening of the alveolar wall. The total lung injury score per mouse = [(20×A) + (14×B) + (7×C) + (7×D) +(2×E)]/(number of fields×100). Immunohistochemical staining was performed using antibodies against MPO. Photographs were taken in a blind fashion at random fields. Representative pictures of lung sections were displayed.

Determination of cell viability by CCK8

The Raw264.7 cells were plated in 96-well plates at 2,000 cells per well. After overnight incubation, different concentration of PGLYRP1-Fc was added to the cells and then cultured for 24 hours. Four wells for each dose were sampled for CCK-8 measurement. Briefly, the culture medium was removed, 90 μL RPMI 1640 basic medium with 10 μL CCK-8 was added, and the cells were cultured in the dark for 1.5 h. OD₄₅₀ was read by Microplate Reader.

Flow-cytometric analysis

1×10⁵ cells were incubated with 1 μg or 5 μg purified proteins on ice for 60 min. Cells were washed and stained with Goat Anti-Mouse IgG (H&L) - Alexa Fluor 488 on ice in dark for 60 min. Cells were washed with 1% FBS in PBS and fluorescence was detected by flow cytometry using Cytoflex (Beckman Coulter, USA). Data were analyzed by FlowJo and FCS Express.

Transwell assay for neutrophil migration

Neutrophils were obtained 7 h after intraperitoneal injection of 1 ml of 3% thioglycolate, as the reported study.⁵⁸ The harvested cells were washed and incubated in RPMI-1640 medium at 37°C for 1 h in 250 mL flasks (Corning). Nonadherent cells containing 80–90% neutrophils were recovered.

Isolated murine neutrophils were resuspended in serum-free RPMI-1640 medium containing 1% P/S at 10⁶ cells/mL. 100 μL cells were added to the upper well of each Transwell chamber (5 μm pore size, Corning) placed in a 24-well plate, and 300 μL serum-freed cell supernatant from the pretreated primary macrophages were added to the lower well as previously described.⁵⁹ RPMI-1640 medium alone was used as a negative control. 10% FBS group was used as a positive control.⁶⁰ After incubation for 4 h, the upper compartment of the Transwell was removed and the lower well was placed in the 24-well plate was taken photo under microscopy. Finally, the medium of the lower well was collected, centrifuged, resuspended in 100 μL PBS and counted with a hemocytometer.

Assay for bacterial killing by cells

1×10⁵ cells were cultured in a 96-well plate with RPMI-1640 and 10% FBS medium without P/S and treated with different doses of fusion proteins for the indicated time. 1 × 10⁵ CFU of luminescent E.coli was added and centrifuged at 250 g at room temperature for 20 min. After incubating samples for 90 min at 37°C, D-Luciferin was added and reacted for 2 min. The luminescence of each well was determined using a Microplate Reader (Tecan, Switzerland).

RNA isolation and real-time PCR

Total RNA was extracted from cells using Trizol reagent according to the manufacturer (Sigma-Aldrich, St.Louis, MO, USA) and then reversely transcribed to cDNA by HiScript II Q RT SuperMix kit (Vazyme, Nanjing, China). According to the manufacturer’s protocol, real-time PCR was performed by the SYBR Green I fluorescent dye (Vazyme, Nanjing, China). GAPDH was an invariant control, and mRNA levels were expressed as fold changes after normalizing to GAPDH. The primer sequences used for murine macrophage can be found in below table.

Primers used for the determination of mRNA expression levels in murine macrophages

Gene	Forward sequence	Reverse sequence
CCL2	5′- AGGTGTCCCAAAGAAGCTGTA-3′	5′- ATGTCTGGACCCATTCCTTCT-3′
CD206	5′-AAACACAGACTGACCCTTCCC-3′	5′-GTTAGTGTACCGCACCCTCC-3′
CXCL1	5′- CCACACTCAAGAATGGTCGC-3′	5′- TCTCCGTTACTTGGGGACAC-3′
CXCL2	5′- GAGCTTGAGTGTGACGCCCCCAGG-3′	5′- GTTAGCCTTGCCTTTGTTCAGTATC-3′
iNOS	5′- CACCTTGGAGTTCACCCAGT-3′	5′- ACCACTCGTACTTGGGATGC-3′
IL-6	5′- CCTGTCTATACCACTTCAC-3′	5′- AATCAGAATTGCCATTGC-3′
IL-10	5′- CCAAGCCTTATCGGAAATGA-3′	5′- TCACTCTTCACCTGCTCCAC-3′
TNF-α	5′- CTGTGAAGGGAATGGGTGTT-3′	5′- GGTCACTGTCCCAGCATCTT-3′
Trem1	5′- ATAAATGGGACAGATGCT-3′	5′- TGACAATGAATAAGATGATGAA-3′
GAPDH	5′-CTTCTTTTGCGTCGCCAGCCGA-3′	5′- ACCAGGCGCCCAATACGACCAA-3′

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Western blot analyses

Tissue and cells were lysed by lysis buffer containing proteinase and phosphatase inhibitors, and the protein concentration was quantified by BCA assay kit. The protein was boiled in the SDS sample buffer. Equal amounts of protein were separated by SDS-PAGE, then transferred onto a nitrocellulose membrane (Millipore). The membranes were incubated at 4°C overnight with primary antibodies against AKT, p65, phosphorylated-AKT, and phosphorylated-p65. β-actin used for loading control was detected with an anti-β-actin antibody. The matched fluorescein-linked secondary antibodies were used to visualize proteins by incubation at room temperature for 1 h. The membranes were scanned with a Gel imaging system (Tanon) and quantified by Image J.

Immunofluorescence analysis

Peritoneal macrophages were cultured in a 24-well plate and given corresponding reagents for the indicated time. Cells were then fixed with 4% PFA for 30 min at 37°C. After that, cells were permeabilized with PBS-T (0.1% Triton X-100 dissolved in PBS) for 10 min and blocked with 4% BSA in PBS. Next, cells were incubated with NF-κB antibody (1:100 dilution) overnight at 4°C, followed by staining with FITC-labeled Goat Anti-Rabbit IgG (1:200 dilution, Jackson ImmunoResearch Inc, West Grove, PA, USA) for 2 h. Finally, cells were stained for DAPI in dark for 5 min and the fluorescence was observed by microscope.

ELISA assay

Cytokines including IL-6, IL-10, TNF-α, and CXCL2 in the cell supernatants of primary macrophages, BALF, and lung homogenate were measured by ELISA kits (Lianke, Hangzhou).

Quantification and statistical analysis

Data were expressed as mean±SD or mean±SEM, and the differences between groups were analyzed by Prism 5.0 (Graph pad Software Inc., San Diego, CA). Survival analysis was determined by a log-rank test. The statistical analysis of the two-group comparison was assessed by Student t-test, while the comparison of more than two groups was determined by one-way analysis of variance (ANOVA). P＜0.05 was considered as a significance.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (81773618).

Author contributions

Conceptualization, Y.J. and W.H.; Methodology, Y.J., W.H., and J.J.L.; Investigation, Y.J., S.R., L.Y.S., and S.Y.W.; Writing – Original Draft, Y.J.; Writing –Review & Editing, Y.J. and L.L.J.; Funding Acquisition, Y.Y., W.H., and J.J.L.; Resources, Y.Y., B.Y.M., and W.H.,; Supervision, Y.Y., B.M.Y., and J.J.L.

Declaration of interests

The authors declare no competing interests.

Published: April 14, 2023

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2023.106653.

Contributor Information

Jingjing Li, Email: lijj@sjtu.edu.cn.

Yan Yu, Email: yanyu@sjtu.edu.cn.

BuYong Ma, Email: mabuyong@sjtu.edu.cn.

Supplemental information

Document S1. Figures S1–S11

mmc1.pdf^{(772KB, pdf)}

Data and code availability

•
All data are included in the published article and the supplemental information files are available from the lead contact upon request.
•
This paper does not report the original code.
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S11

mmc1.pdf^{(772KB, pdf)}

Data Availability Statement

•
All data are included in the published article and the supplemental information files are available from the lead contact upon request.
•
This paper does not report the original code.
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

[bib1] 1.Thompson B.T., Chambers R.C., Liu K.D. Acute respiratory distress syndrome. N. Engl. J. Med. 2017;377:1904–1905. doi: 10.1056/NEJMc1711824. [DOI] [PubMed] [Google Scholar]

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PERMALINK

PGLYRP1-mIgG2a-Fc inhibits macrophage activation via AKT/NF-κB signaling and protects against fatal lung injury during bacterial infection

Yan Jia

Shan Ren

Luyao Song

Siyi Wang

Wei Han

Jingjing Li

Yan Yu

BuYong Ma

Summary

Graphical abstract

Highlights

Introduction

Results

Construction, production, and functions of Fc-fusion proteins

Figure 1.

PGLYRP1-Fc protects against lethal acute bacterial pneumonia

Figure 2.

PGLYRP1-Fc improves lung inflammation and injury without affecting the bacterial burden

Figure 3.

PGLYRP1-Fc ameliorates pathological lung damage during bacterial lung infection

Figure 4.

PGLYRP1-Fc regulates LPS-induced inflammatory response in macrophages and inhibits activated-macrophages induced-neutrophil migration

Figure 5.

PGLYRP1-Fc suppresses inflammatory response in macrophages via AKT mediated NF-κB pathway

Figure 6.

PGLYRP1-Fc reduces lung inflammation via AKT/NF-κB in mice

Figure 7.

PGLYRP1-Fc impairs the bactericide and phagocytosis of macrophages and does not affect the bactericidal ability of neutrophils

Figure 8.

Discussion

Limitations of the study

STAR★Methods

Key resources table

Resource availability

Lead contact

Materials availability

Experimental models and subject details

Animals

Cell culture

Bacteria culture

Method details

Construction, purification, and characterization of Fc fusion proteins

Bacteria binding assay

LPS binding assay

Cell culture and treatment

The opsonophagocytic killing of E.coli

Macrophage phagocytosis assay

Bacteria infection-induced ARDS model

Bacterial burden

BALF cell count and differentiation

Lung histopathology and immunohistochemistry

Determination of cell viability by CCK8

Flow-cytometric analysis

Transwell assay for neutrophil migration

Assay for bacterial killing by cells

RNA isolation and real-time PCR

Western blot analyses

Immunofluorescence analysis

ELISA assay

Quantification and statistical analysis

Acknowledgments

Author contributions

Declaration of interests

Footnotes

Contributor Information

Supplemental information

Data and code availability

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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