Adoptive cell transfer of piezo-activated macrophage rescues immunosuppressed rodents from life-threating bacterial infections

Liu, Xiaoyi; Xu, Wenxiu; Feng, Junkun; Wang, Ying; Li, Kai; Chen, Yi; Wang, Wenjun; Zhao, Weiwei; Ge, Shaohua; Li, Jianhua

doi:10.1038/s41467-025-56460-2

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Published: 04 February 2025

Adoptive cell transfer of piezo-activated macrophage rescues immunosuppressed rodents from life-threating bacterial infections

Xiaoyi Liu¹,
Wenxiu Xu¹,
Junkun Feng¹,
Ying Wang¹,
Kai Li¹,
Yi Chen¹,
Wenjun Wang¹,
Weiwei Zhao¹,
Shaohua Ge ORCID: orcid.org/0000-0003-3821-5480¹ &
…
Jianhua Li ORCID: orcid.org/0000-0001-5227-8627¹

Nature Communications volume 16, Article number: 1363 (2025) Cite this article

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Abstract

Bacterial infections pose a significant threat to human health. Catalytic antibacterial nanoparticles that generate reactive oxygen species (ROS) are emerging, as a promising therapeutic approach in treating bacterial infection by boosting the innate immune defenses. However, the interaction between innate immune cells and these catalytic nanoparticles remains poorly understood. Here, by using rodent models of bacterial infection, we test the antimicrobial properties of ultrasound-responsive piezo-catalytic nanoparticles (piezoNP). We show that piezoNPs strongly interact with macrophages within subcutaneous abscesses caused by Staphylococcus aureus (S. aureus) infections, and demonstrate that this interaction enhances the macrophage-mediated antibacterial phagocytosis and killing activity through intracellular piezocatalysis. Moreover, we test the use of these piezo-activated macrophages (piezoMϕ) as adoptive cell therapy (ACT) for treating various immunosuppressive bacterial infections, including sepsis, pneumonia and peritonitis. Our study thus highlights the potential application of catalytic nanoparticles as a promising alternative to conventional infection treatment to effectively modulate the innate immune responses and to engineer macrophages for immunotherapy purposes.

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Introduction

Bacterial infections cause millions of deaths worldwide each year, significantly contributing to the global burden of disease¹. Bacterial pathogens weaken the innate immune response by producing various virulence factors that counteract the defense mechanisms of professional phagocytes^2,3, thereby creating an immunosuppressive microenvironment that sustains infection and the associated inflammation⁴. Although antibiotic therapy has been the standard treatment for the past century, it offers limited benefits in repairing the immunosuppressive microenvironment or regenerating innate immune cells, and it may even lead to the development of uncontrollable drug-resistant strains⁵.

Antibacterial nanomaterials are gaining attention because of their versatile physiochemical properties, absence of drug resistance, and the capacity to regulate the immune system^6,7,8. For example, catalytic nanoparticles can eliminate bacteria by producing ROS^9,10,11. Meanwhile, immune cells at the infection location may internalize these nanoparticles. Recent studies indicate that macrophages are the primary innate immune cells involved in the recognition of these therapeutic nanoparticles^12,13. Macrophages are important innate immune cells and the first line of defense when combating bacterial infection^14,15,16,17. However, little is known about how catalytic nanoparticles interact with macrophages during the clearance of infection^18,19.

Macrophages are increasingly being recognized as promising targets for cell immunotherapy in the treatment of infectious diseases and cancer^20,21,22. Due to the intrinsic phagocytosis and immune stimulation of M1 macrophages, strategies based on engineered macrophages or adoptive cell therapy (ACT) have been proposed^{23,24,25,26,27}. Beyond pro-inflammatory factors such as interferon-gamma (IFN-γ), reactive oxygen species (ROS), a powerful initiator and enhancer of the immune response, exhibits great potential in directing macrophage polarization towards the M1 phenotype²⁸. For instance, cancer immunotherapy has been achieved by using nanocatalysts to produce intracellular ROS from endogenous H₂O₂, thereby inducing M1 polarization of macrophages²⁹. Consequently, it is anticipated that catalytic nanoparticles could enable in situ ROS generation and modulate the innate response of macrophages in the battle against infection.

In this work, we employ piezocatalytic nanoparticles (piezoNP) composed of a BaTiO₃@Au metal-piezoelectric heterostructure as ultrasound (US)-responsive nanocatalytic ROS generating agents. We show that piezoNPs achieve effective bacteria clearance upon US irradiation. We demonstrate that piezoNPs are spontaneously identified by immune cells within Staphylococcus aureus (S. aureus) abscess and strongly associate with macrophages. The piezoMϕ exhibits enhanced phagocytosis and killing of S. aureus, demonstrating US-boosted antibacterial capabilities. We further demonstrate the effectiveness of piezoMϕ as ACT in treating immunosuppressive infection conditions, including sepsis, pneumonia, and peritonitis. Our work highlights the importance of innate immune activation in treating bacterial infection with catalytic nanoparticles, providing insight for broader nanoparticle-mediated immune cell therapy.

Results

Piezocatalytic generation of ROS

The piezoNPs (BaTiO₃@Au) were prepared by pieodeposition of Au on BaTiO₃ (BTO) nanoparticles (Fig. 1a)^30,31,32. Typical transmission electron microscopy (TEM) images (Fig. 1b) and energy dispersive X-ray spectroscopy (EDS) element mapping (Fig. 1c) show that Au NPs (average diameter of 5 nm) grew on the surface of BTO, which had an average size of 50 nm. The (111) facets of Au, corresponding to 0.23 nm lattice fringes, and the (100) facets of BTO, corresponding to 0.4 nm lattice fringes, are clearly observed in Fig. 1c. The metal-piezoelectric heterostructure can be clearly observed in Fig. 1d. X-ray diffraction (XRD) analysis (Fig. 1e) reveals the crystallization of Au and typical tetragonal BTO diffraction peaks, indicating that BTO@Au retains its inherent ferroelectric/piezoelectric phase. The piezoelectric discharge from piezoNPs was then measured by imposing the nanoparticle-mounted electrode under a periodic US irradiation. As seen in Fig. 1f, an electrical signal from piezoNPs greater than that of the pristine BTO NPs was observed, suggesting that the metal-enhanced charge separation and migration is beneficial for subsequent piezocatalysis of BTO@Au nanoparticles in aqueous solution^33,34,35. The total ROS generation under US irradiation was significantly higher for the piezoNPs (Fig. 1g), and the ROS production was influenced by the applied US power and irradiation time (Supplementary Fig. 1). Under the action of ultrasonic vibration, BTO@Au NPs are stressed and polarized to generate piezoelectric charges, which further react with the dissolved oxygen and water molecules to produce ROS, including superoxide radical (⋅O₂⁻) and hydroxyl free radical (⋅OH)³² (Fig. 1h and Supplementary Fig. 2). As a result, the obtained piezoNPs have efficient and controlled US-responsive piezocatalytic ROS production.

**Fig. 1: Characterization of piezoNPs and US-controlled piezocatalysis.**

Piezocatalytic inhibition of S. aureus

Next, we investigate the bacteria inhibition effect of piezoNPs subjected to US irradiation. We chose S. aureus as a model pathogen^36,37, which is a leading cause of a vast spectrum of infections, from minor soft-tissue infections to life-threatening bacteremia, sepsis, or endocarditis³⁸. The nanoparticles were dispersed into bacteria suspension and the mixture was then challenged with US at 1 W cm⁻². Figure 2a depicts the ultrasonic time-dependent survival rate of S. aureus; after 30 min of treatment, the piezoNP+US group’s survival rate was less than 1%, while the survival rates of the other treatment groups remained above 90% (relevant plate counting images displayed in Supplementary Fig. 3). The ROS-dosing dependent deactivation of bacteria was also revealed by varying piezoNPs concentration (Supplementary Fig. 4). After US treatment, the mixture quickly sank to the bottom (Supplementary Fig. 5), indicating that piezocatalytic treatment damaged the bacterial colloidal stability. Bacterial hyperpolarization status and membrane potential (Supplementary Fig. 6) were found to be significantly lower after piezocatalytic treatment, indicating that the integrity of the bacterial cell membrane had been compromised. The piezoNPs were bounded to the S. aureus membrane in TEM images, that piezoNPs+US treated bacteria revealed a broken membrane (Fig. 2b). Consequently, bacteria treated with piezoNPs+US showed higher levels of protein leakage, protein oxidation, and lower intracellular ATP activity (Supplementary Fig. 7). The capacity of S. aureus to invade human cells was eventually inhibited after piezocatalytic treatment (Supplementary Fig. 8). The results show bacterial cell membrane-bound piezocatalysis can produce localized oxidative stress, leading to altered membrane integrity, aberrant energy metabolism, thus ultimately contributing to the inhibition of bacterial infection (Fig. 2c).

A rat subcutaneous abscess model was then used to investigate the piezocatalytic inhibition of S. aureus in vivo (Fig. 2d). A common feature of S. aureus infections is the formation of abscesses, which contain bacterial communities and reservoirs of host immune cells, providing a confined lesion region to explore how nanoparticles participate in the bacteria-immune cell interaction. For optimal action, piezoNPs were injected directly into the abscess, and ultrasonic insonation from a commercial ultrasonic physiotherapy instrument (Supplementary Fig. 9) was applied perpendicularly to the skin (Fig. 2e). After four days of treatment, a significant reduction in the abscess area (over 83.02% decrease in size, Fig. 2g), as well as the inactivation of S. aureus (99.99% reduction in bacteria burden, Fig. 2h), were clearly observed in the piezoNPs+US group (Fig. 2f). Hematoxylin-eosin (H&E) staining images (Fig. 2i and Supplementary Fig. 10) demonstrate that the Control, US and piezoNPs treated groups all exhibited inflammation with significant inflammatory cell infiltration. Pro-inflammatory cytokines tumor necrosis factor (TNF), interleukin (IL)-1, and IL-6 appeared in the serum further verifying the local infection-induced systemic inflammation. Four days after piezocatalytic treatment, the release of those pro-inflammatory mediators was dramatically reduced to normal levels (Fig. 2j, detailed bar chart Supplementary Fig. 11). It is worth noting that the US dose applied in vivo (1 W cm⁻², 3 min) was weaker than that used in in vitro antibacterial study (1 W cm⁻², 30 min), the superior antibacterial performance in vivo inspired us to investigate the immunological effect of piezoNPs on host innate defense.

Association of piezoNPs with macrophages in S. aureus abscess

The innate immune cells involved in combating S. aureus infection mainly include neutrophils and macrophages³. Macrophages have an essential function in the initial clearance of S. aureus and control the flow of neutrophils and monocytes to the infection site. However, S. aureus can evade these innate immune responses, leading to the formation of an abscess to maintain the infection nidus (Fig. 3a). Therefore, we next wanted to assess whether piezoNPs within the abscess interact with these innate immune cells and contribute to bacterial clearance.

**Fig. 3: Association of piezoNPs with macrophages at the infection site.**

Flow cytometry of signal-cell suspension isolated from the abscessed tissue was performed to examine the interaction between CY5-labeled piezoNPs (Supplementary Fig. 12), HIS48⁺ neutrophils, and CD11b/c⁺ macrophages after piezoNPs injection. The results show that in all living cells (Supplementary Fig. 13), the frequencies of piezoNPs⁺ CD11b/c⁺ macrophages were 10.4%, 12.1%, 13.3%, and 15.8% at 1, 8, 16, and 24 h after injection, respectively, without US treatment; whereas NPs⁺ CD11b/c⁺ macrophages varied from 16.0% to 41.3% after US treatment (Fig. 3b and Supplementary Fig. 14a), which indicates that the macrophages in abscess had a high interaction with the injected piezoNPs. After US irradiation, the association rate of piezoNPs with macrophages increased, which may be related to the restoration of macrophage function initially repressed by active S. aureus. The frequencies of piezoNPs⁺ HIS48⁺ neutrophils at 24 h after NPs injection were only 11.3% without US and 17.2% with US, respectively (Fig. 3c and Supplementary Fig. 14b). In addition, the ratio of macrophages or neutrophils revealed slight change during piezocatalytic treatment (Supplementary Fig. 15a). The results suggest that macrophages show a higher propensity for engaging with piezoNPs, which is further identified by the co-localization of piezoNPs and macrophages/neutrophils in the abscess tissue section (Fig. 3d and Supplementary Fig. 15b–d). In regards to the dynamic co-localization analysis, as shown in Fig. 3e, f, the overlap of piezoNPs with the macrophages steadily increased over the course of 24 h after piezoNPs injection, and the piezoNPs+US group demonstrated a higher NPs-macrophage association.

We next verify the role of macrophages/piezoNPs interaction in piezocatalytic clearance of infection. Clodronate liposomes (CL) or anti-lymphocyte antigen 6 complex locus G (anti-Ly6G) antibody were given intraperitoneally to deplete the macrophages or neutrophils in vivo prior to the piezocatalytic treatment, respectively (Fig. 3g). In the presence of piezo-treatment (piezoNPs+US), the average abscess size in the neutrophil-depleted group reached to 14.19 mm², which is much smaller than that in the macrophage-depleted group (74.92 mm²) (Fig. 3h, i). When macrophages were depleted, the piezo-treated infection showed only an 11.87% decrease in abscess size compared to the non-piezo-treated group, indicating no significant difference between the groups. Conversely, when neutrophils were depleted, the piezo-treated infection showed a 74.44% decrease in abscess size compared to the non-piezo-treated group (Fig. 3h, i), highlighting a more significant role of piezoNPs in the presence of macrophages rather than neutrophils. Therefore, the superior antibacterial performance in vivo should be attributed to the direct piezocatalytic deactivation of bacteria (Fig. 1), and the piezo-activation of macrophage (Fig. 2), the latter of which is suggested to play a crucial part in the clearance of S. aureus infection.

Macrophages primed with piezoNPs exhibit US-boosted antibacterial activities

Next, we wanted to clarify how the piezoNPs/macrophage interaction participate to the antibacterial mechanisms. One major antibacterial mechanism of phagocytes is the production of ROS via activating host inflammatory signaling. However, S. aureus have evolved mechanisms to withstand ROS and release other virulence factors to attenuate the innate immune responses^5,39,40. We hypothesize that piezoNPs uptaken by the macrophages can initiate US-driven intracellular piezocatalysis, thereby enhancing a robust antibacterial and pro-inflammatory response to bacterial invasion. To better understand the antibacterial mechanism underlying piezoNPs/macrophage interaction, piezoNPs were incubated with rat bone marrow-derived macrophages (BMDMs, Supplementary Fig. 16) to obtain the piezoNPs-primed macrophage (piezoMϕ). The piezoMϕ irradiated with US were then compared to the pristine BMDMs in terms of their transcriptome profiles following exposure to S. aureus. Genes Ontology (GO) analysis revealed that the majority of differentially expressed genes in the US-activated piezoMϕ were enriched in inflammatory response signaling pathways (Supplementary Fig. 17a). The top 20 KEGG enrichment analysis revealed that upregulated genes were enriched in innate immune response-associated signaling pathways, such as TNF, NF-ĸB (nuclear factor-ĸB), IL-17, C-type lectin receptors, and Toll-like receptors signaling pathways (Fig. 4a). This suggests that piezo-activation can effectively strengthen innate immune function during bacterial infection^41,42. In both primary Mϕ and activated piezoMϕ, genes related to the innate immune response were significantly upregulated upon pathogen contact (Fig. 4b). Hierarchical cluster analysis of innate immune response genes indicated a notable upregulation of genes associated with NF-κB signaling (Supplementary Fig. 17b), a key regulator of the host inflammatory response against bacterial pathogens⁴³. We further analyzed the regulated genes associated with phagocytosis (Fig. 4c) and ROS signaling (Supplementary Fig. 17c). Phagocytosis-related genes including cytokines (e.g., IL1a/b, IL10), chemokines (CCL3, CCL4, CXCL1, CXCL2, CXCL3), and inducible nitric oxide synthase (iNOS or NOS2), a major component of the antimicrobial effector machinery responsible for producing bactericidal nitric oxide (NO), were significantly upregulated in US-activated piezoMϕ⁴⁴.

**Fig. 4: Macrophages primed with piezoNPs exhibit ultrasound-boosted antibacterial activities in vitro.**

To confirm US-boosted antibacterial activity of piezoMϕ, cells were challenged with S. aureus in vitro. According to flow cytometry results (Fig. 4d and Supplementary Fig. 18), US-activated piezoMϕ demonstrated powerful phagocytosis activity with more intracellular pHrodo-S. aureus found in the piezoNPs⁺ cells (influenced by piezoNPs concentrations (Supplementary Fig. 19) and the incubation time (Supplementary Fig. 20). Furthermore, during 60 min of bacterial infection, the phagocytized bacteria resulted in a considerable decrease of viability in activated piezoMϕ as indicated by the intracellular live bacteria counting results (Fig. 4e, f). US-activated piezoMϕ can produce transient intracellular ROS (Fig. 4g), which is considered to benefit intercellular bacteria killing. A well-controlled transient ROS was found to cause relatively low cytotoxicity (Supplementary Fig. 21). Then, we selected Rosup to stimulate intracellular ROS production as a positive control group (Supplementary Fig. 22). The phagocytosis and bacteria killing of US-activated piezoMϕ were comparable to that of positive control (Supplementary Fig. 23), indicating the significant role of ROS-driven immune response during piezo-activation. The activation of NF-ĸB signaling and subsequent proinflammatory pathways is well recognized in macrophage-mediated bacteria clearance. This was confirmed in the activated piezoMϕ group, as evidenced by NF-ĸB p65 phosphorylation (Fig. 4h and Supplementary Fig. 24), mRNA expression of related genes (Supplementary Fig. 25), and increased production of TNF (Fig. 4i), IL-1β (Fig. 4j), and higher release of NO (Fig. 4k). Collectively, the piezocatalysis within macrophages enhances intracellular ROS levels, stimulates phagocytosis activity, and activates proinflammatory signaling pathways and innate immune response. These mechanisms contribute to US-boosted antibacterial activities of piezoNPs-primed macrophages (Fig. 4l).

Piezo-activated macrophages for adoptive cell immunotherapy

The above results motivated us to explore the potential of using activated piezoMϕ as ACT therapy of infectious diseases. In particular, BMDMs were incubated with piezoNPs to form piezoMϕ, which were subsequently activated by US irradiation before ACT (Fig. 5a). High cell viability could be well maintained after piezo-activation in vitro (Fig. 5b) and following cell transfer in vivo (Supplementary Fig. 26), thereby ensuring the effectiveness and efficiency of ACT in treating infections.

**Fig. 5: US-activated piezoMϕ for adoptive cell immunotherapy of *S. aureus-*induced abscess and sepsis infection with immunosuppression.**

For ACT therapy of S. aureus infection, macrophages were transferred by either local delivery to a chronic skin abscess model or intravenously injection to a systemic sepsis infection model. The S. aureus abscess model was developed in rats with cyclophosphamide (CY)-induced immunosuppression to mimic patients with chronic infection. The abscess size significantly reduced in the group treated with activated piezoMϕ after 24 h of cell therapy compared to those treated with local injections of PBS, Mϕ, or piezoMϕ (Fig. 5c, d), which suggests that the in vitro activated piezoMϕ maintained their antibacterial activities in vivo after cell transfer.

Every year, sepsis affects 30 million people worldwide and is an extremely serious infection⁴⁵. Patients with sepsis who survive the early cytokine-mediated hyperinflammatory phase may still be at great risk for infection in the later immune-suppressive phase due to immune cell malfunction and apoptosis. Then, using CY to simulate an immunosuppressed state in sepsis patients, we examined the therapeutic effects of piezoMϕ (BMDMs isolated from mice, Supplementary Fig. 27) in a sepsis mouse model. Specifically, CY-treated mice were intravenously (i.v.) infected with S. aureus and subsequently treated with PBS, Mϕ, piezoMϕ or activated piezoMϕ (Fig. 5e). In comparison to the other groups, the survival rate of US-activated piezoMϕ group (87.5%) was noticeably higher (Fig. 5f). After 20 days of ACT, the body weight of the surviving mice fully returned to that of healthy mice of the same age (Fig. 5g). We further assessed bacterial CFUs in blood and organs, as mortality in immunosuppressed sepsis is associated with overwhelming pathogens in these tissues. Following treatment with US-activated piezoMϕ, the bacterial burden in all organs and blood considerably decreased 24 h after cell transfer (Fig. 5h). After 20 days of treatment, very few bacteria remained in the organs. The activated piezoMϕ therapy efficiently restored white blood cells (WBCs) and lymphocytes (LYMs) level in the septic mice to normal levels after 20 days (Fig. 5i, j), demonstrating a significantly greater therapeutic capacity to eliminate systemic pathogens in vivo.

Given these encouraging findings, we tested the effectiveness of the piezoMϕ-based ACT therapy for two significant infection models: Pseudomonas aeruginosa (P. aeruginosa) pneumonia and methicillin-resistant Staphylococcus aureus (MRSA) peritonitis⁴⁶. To simulate hospital-acquired pneumonia, P. aeruginosa was inhaled by CY-treated C57 mice to establish a pneumonia model (Fig. 6a). With a survival rate of 88.0% after 7 days, US-activated piezoMϕ treatment successfully rescued the sick mice, while all animals were dead within 2 days in the other groups (Fig. 6b). As indicated by the in vivo live cell tracking, the piezoMϕ can retain their viability within 7 days, ensuring their long-term antibacterial activity after ACT (Supplementary Fig. 28a–c). The bacterial burden in the lungs of activated piezoMϕ-treated mice showed a reduction of almost four orders of magnitude in CFUs (Fig. 6c). Additionally, bronchoalveolar lavage fluid (BALF) was collected and examined to measure the secreted pro-inflammatory cytokines. Notably, IL-6 and TNF levels within BALF significantly reduced in activated piezoMϕ group (Fig. 6d, e). Lung histology sections from the infected mice displayed extensive regions of significant granulocytic infiltration, severe bleeding, and alveolar edema, whereas these features were barely noticeable in mice treated with US-activated piezoMϕ (Fig. 6f).

**Fig. 6: ACT with piezoMϕ for *P. aeruginosa-*induced lung infection.**

The distribution of Mϕ in mice was then examined using IVIS imaging of DIR-labeled cells in mice with induced pneumonia. The majority of the transferred cells were found to be accumulated in liver and lungs across all treatment groups (Fig. 6g and Supplementary Figs. 29 and 30), consistent with previously reported patterns of adoptive macrophage distribution²⁴. However, the activated piezoMϕ significantly increased the overall cell intensity in these organs compared to Mϕ and piezoMϕ. Figure 6h demonstrates the activated piezoMϕ quickly accumulated in infected lung tissues during 24 h and diminished by 72 h as infection cleared. The lung/heart cell distribution ratio was higher than the lung/other organs ratio, indicating that activated piezoMϕ quickly entered the infected lung from circulation, peaking at 24 h (Supplementary Figs. 29–31). Since the infected tissues with inflammation have higher vascular permeability^47,48, we anticipate that the activated piezoMϕ may have an enhanced cell migration activity, allowing them to accumulate in infected lung tissues through permeable blood vessel walls (Fig. 6i). To verify this hypothesis, we tested cell migration with or without the presence of bacteria by a transwell assay (Fig. 6j). The results showed that activated piezoMϕ had a much stronger migration ability, which was further enhanced under bacterial infection or inflammatory conditions (Fig. 6k, l). The activation of migration-related genes in the activated piezoMϕ (Supplementary Fig. 32) further supported this point. In addition, fluorescence imaging of the infected lung tissue section after ACT treatment revealed that the piezoMϕ were located inside and beside the CD31-positive regions, where they could engage with the bacteria (Supplementary Fig. 33). The elevated cell migration ability towards the infection site can be advantageous to ACT and the following infection clearance process in vivo.

The increasing prevalence of multi-drug-resistant infections has raised significant concerns about the clinical efficacy of antibiotics, posing a serious threat to human health⁴⁹. Lastly, the adoptive piezoMϕ therapy for a MRSA peritonitis was demonstrated in mice (Fig. 7a). As shown in Fig. 7b, the survival rate of peritonitis mice in the activated piezoMϕ group was 100%, whereas all mice died within 2 days post infection in the Mϕ or piezoMϕ treated groups. Additionally, the activated piezoMϕ eliminated more than 99.9% pathogens in all organs by day 7 post infection (Fig. 7c). Again, the piezoMϕ can retain 50% of their viability after 24 h, ensuring their long-term antibacterial activity after ACT (Supplementary Fig. 28d–f). The in vivo antimicrobial studies demonstrated the potency of activated piezoMϕ in treating a broad spectrum of infections.

**Fig. 7: Treatment of MRSA-induced peritonitis infection.**

We also verified the piezoMϕ based ACT strategy on infected animals without CY-induced immunosuppression. The results show that the activated piezoMϕ could rescue the infected immune-complete animals and reduce the bacterial load by 2–3 orders of magnitude in both MRSA-induced peritonitis infection and P. aeruginosa-induced lung infection (Supplementary Fig. 34). Furthermore, the activated piezoMϕ ACT therapy demonstrated superior efficacy in bacterial elimination and survival rates compared to standard antibiotic treatments, including Vancomycin and the Piperacillin/Tazobactam combination (Supplementary Fig. 35).

In comparison to traditional macrophage activation methods, such as interferon-γ (IFN-γ) or lipopolysaccharide, the piezo-activated Mϕ ACT treatment demonstrated enhanced efficacy in bacterial eradication and improved survival rates (Supplementary Fig. 36). Furthermore, the localized abscess lesions shown in Fig. 2 allow the injected piezoNPs to directly interact with bacteria and macrophages. However, in more widespread infections such as sepsis, pneumonia, and peritonitis, it is challenging to deliver piezoNPs specifically to activate macrophages effectively (Supplementary Fig. 37). Therefore, the proposed piezoMϕ ACT therapy may address the limitations of current bacterial infection treatments, overcoming challenges such as antibiotic resistance, varying host immune states and targeted treatment^50,51.

It should be noted that the harvest of autologous macrophages might be a challenge due to the weak conditions of patients and the tedious preparation of engineered cells. However, recently-developed techniques such as bioreactor-based mass production of macrophages from human induced pluripotent stem cells (iPSC) may provide “off-the-shelf” autologous/allogeneic cell source⁵². Another potential limitation of this work is that BaTiO₃@Au piezoNPs are inorganic materials considered to be non-degradable and bio-inert, as shown in the stability test in cells (Supplementary Fig. 38)⁵³.

Discussion

In summary, we have demonstrated the crucial role of macrophages in fighting against bacterial infection in conjunction with ROS catalytic nanoparticles. We developed a macrophages-based ACT strategy using piezoNPs-mediated activation to enhance antibacterial phagocytosis and killing ability. Our results prove that the adoptive transfer of piezoMϕ effectively rescues immunosuppressed rodents from sepsis, pneumonia, and MRSA peritonitis.

Although the mechanism of piezoelectric catalysis in producing superoxide and hydroxyl radicals is relatively well understood, the impact of the specific types of free radicals generated intracellularly on the antibacterial activity of macrophages remains unclear. In the future, more catalytic nanoparticles with unique properties, such as biodegradability, responsiveness, or drug-loading ability, should be explored, so that the proposed strategy can be adopted for infections in various clinical settings with diverse needs. Engineering macrophages with other functional catalytic nanomaterials should also be expected in future studies.

In addition, by taking advantage of the tissue penetration of US and targeting ability of nanoparticles, in situ engineering and activation of macrophages at the infection site may also be achieved for on-demand and remote treatment of deep-tissue infections. Overall, this work supports using ROS catalytic nanoparticles to modulate bacterial-immune interactions. Piezo-activated macrophages provide a promising cell source for curing patients with life-threatening bacterial infections.

Methods

Materials

BaTiO₃ nanoparticles purchased from Hefei Kemike Biochemical Technology Co., Ltd, (3-Mercaptopropy I) trimethoxy silane (Heowns, 4420-74-0), HAuCl₄ (ZGSJW, 16903-35-8), DiL cell membrane orange fluorescence response (Solarbio, D8700), 2, 7-Dichlorodihydrofluorescein diacetate (AGSJW, 16903-35-8), Triton X-100 (Solarbio, T8200), CCK-8 (Biosharp, 521942), pHrodo^TM Green STP ester (Thermo Fisher Scientific, P35369), RPMI 1640 (BININD, 01-100-1A), FBS (BIOIND, 04-001-1ACS), Collagenase I (Solarbio, C8140), Dispase II (Solarbio, D6430), Elastase (Shanghai yuanye Bio-Technology Co., Ltd, S10165), CY5-PEG-SH (Xi’an Ruixi Biological Technology Co., Ltd), Clodronate Liposomes (Shanghi SunLipo BioTech Co. Ltd, SN-ML-E010), anti-Ly6G antibody (Bio X Cell, BE0075-25), Cyclophosphamide (HEOWNS, C-0232450) FITC Anti-rat CD11b/c Antibody (Biolegend, 201805), Granulocyte Marker Monoclonal Antibody (Thermo Fisher Scientific, 13-0570-82), APC Anti-mouse F4/80 Recombinant Antibody (Biolegend, 157305), FITC Anti-mouse CD11b (Biolegend, 101205), FITC-HIS48 (eBioscience, 11-0570-82), Anti-CD11b Antibody (abcam, ab1211), Anti-Granulocytes antibody[HIS48] (abcam, ab33760). SYTO™ 9 Green Fluorescent nucleic acid stain (Thermo Fisher Scientific, S34854), Anti-CD31 antibody (abcam ab182981), Propidium iodide bacterial dye (Sigma-Aldrich, 25535-16-4), DiR (Xi’an Ruixi Biological Technology Co.), Anti-NF-κB p65 Abtibody (phospho S536) (abcam, ab76302), Anti-NF-κB p65 (acetyl K310) Antibody (abcam, ab19870), Anti-GAPDH Antibody (abcam, ab181602), Rat TNF ELISA Kit (Proteintech, KE20001), Rat IL-1β ELISA Kit (Proteintech, KE20005), Mouse TNF ELISA Kit (Proteintech, KE100002), Mouse IL-6 ELISA Kit (Proteintech, KE100007). NO detection kit (Solarbio, BC1470).

Animals

Wistar IGS Rats (102) and C57BL/6JNifdc (212) Mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd, China. All animals were raised in the Experimental Animal Center of Shandong University under specific pathogen-free conditions. All animal experiments were approved by the Ethics Committee of Stomatological Hospital of Shandong University (No. 20210605) and performed according to the guidelines. The animal experimental protocols adhered to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. The experimental/control animals were co-housed. The housing conditions for the animals were 12-h light/12-h dark cycle, temperatures of 20–26 °C with 40–70% humidity. For the experiments on rats, male rats, aged six weeks, with a mean weight of 200 ± 20 g were employed. The sex of mice used in the study was male, and the weight was 19–21 g.

Synthesis and characterization of piezoNPs

~100 nm piezoNPs were synthesized by an in situ piezodeposition method according to our previous report³². Briefly, 10 mg of BaTiO₃ NPs were dispersed into 10 mL ethanol. Then 100 μL of (3-Mercaptopropy I) trimethoxy silane was added into the mixture, followed by ultrasonication. The washed particles were then added to 10 mL of 20 mM HAuCl₄ solution and 10 mL of 20% methanol. The pH of the mixture was adjusted to about 9.7 by adding K₂CO₃ solution. The mixture was then treated with ultrasonic vibration (40 kHz, 80 W) at 4 °C.

The crystalline structure of the synthesized piezoNPs was characterized with X-ray diffractometer (XRD, SmartLab 3 kW, Rigaku) using monochromatic Cu Kα radiation (λ = 1.5406 Å, 2θ = 10–90°). The elemental analysis and elemental mapping were conducted with an energy dispersive X-ray spectroscopy (EDS, JEM-2100F, JEOL). The morphology of the samples was observed with high solution transmission electron microscopy (HRTEM, JEM-2100, JEOL).

The piezoelectric discharge signal from piezoNPs was detected on a CHI66E electrochemical workstation using a three-electrode system. The supporting electrolyte is 4 mL 0.5 M Na₂SO₄ solution containing 4 mM ethylenediaminetetraacetic acid disodium salt. 2.5 mg of NPs were dispersed into 300 μL ethanol with 200 μL Nafin added. 20 μL of the mixture was dropped on the surface of disk working electrode and allowed to be dried. Periodic ultrasound (1 W cm⁻²) was applied by using an ultrasonic physiotherapy instrument (WED-100, WELLD, China) and the electric signal was recorded by the electrochemical workstation.

To determine the piezocatalytic generation of ROS, 15 mg of piezoNPs was dispersed in 9 mL 10 μM DCFH-DA solution before treated with ultrasound (2.5 W cm⁻²) in dark condition. 750 μL of the supernatant was sampled and analyzed by a fluorescence spectrophotometer (F-4700, Hitachi) at the excitation wavelength of 504 nm. The produced radicals were also identified by ESR spectra.

Piezocatalytic inactivation of bacteria

Single S. aureus (ATCC 6538) colony growing in logarithmic period was diluted with 0.85% NaCl gradient to 1 × 10⁹ CFU mL⁻¹. 10 mg piezoNPs was dispersed into 1 mL of 1 × 10⁸ CFU mL⁻¹ bacterial suspension in a sterile glass bottle. The mixture was treated with ultrasound (2.5 W cm⁻²) at room temperature in dark. The suspension was collected and diluted to 1 × 10⁴ CFU mL⁻¹ for plate-counting at 37 °C for 24 h. The bacterial cell membrane potential was measured using a Cell Membrane Potential Detection Kit (BB-4110, Bestbio, China). Bacterial membrane potential was also detected by a zeta potential meter (Malvern ZEN3700, UK). The bacterial live/dead staining was conducted according to the manufacture’s protocol and the fluorescent images were taken on a microscope (TH4-200, OLYMPUS). The morphological changes of bacteria were observed by a scanning electron microscopy (Pro G5, Phenome). The internal structure changes of the bacteria were observed by a TEM (Talos F200C, Thermo Fisher).

Piezocatalytic treatment of subcutaneous S. aureus infection

Wistar rats were utilized for this study, with 5 rats assigned to each group and a total of 100 rats involved. Rats were anesthetized with isoflurane (R510-22, RWD, China) before surgery. After shaving and disinfecting with iodophor, a suspension of S. aureus in Luria-Bertani medium (1 × 10⁸ CFU ml⁻¹, 100 μL) was subcutaneously injected into the rat back. Abscess e formed 24 h post infection. The piezoNPs (5 mg mL⁻¹, 100 μL) was then directly injected into the infected abscess. 1 W cm⁻² US was applied to treat the abscess for 3 min and three times a day. Animals were exposure to carbon dioxide to euthanasia. Rat skins were exposed to take photographs of the abscess every day for consecutive 4 days (n = 5). Infected area was calculated using the formula: S = L/2 × W/2 × π, where L and W were the longest and shortest dimensions of the abscesses, respectively. Abscesses were then harvested for histological staining and the bacteria content in abscess tissue lysate was measured by standard plate count methods. Blood was collected into the collection vessels that not coated with heparin. The serum was harvested and stored at −80 °C after the blood was centrifuged at a 1500 × g for 20 min for systemic inflammatory factor analysis.

Association of piezoNPs with innate immune cells in abscess

Abscess tissues from rat were snap-frozen in optimum cutting temperature compound (Tissue-Tek) andsectioned serially (10 μm) using a Leica CM1860 UV cryostat. Sections fixed with cold 4% PFA were stained with CD11b/c antibodies (abcam; ab1211, 1:2000 dilution) and HIS48 antibody (abcam; ab33760, 1:1000 dilution) in immunefluorescence staining using Cy3-conjugated Affinipure Goat Anti-Mouse IgG (Proteintech, 1:100 dilution) and Alexafluor488-conjugated anti-mouse IgM (abcam, 1:1000 dilution). DAPI was used as the counterstain.

Signal-cell suspension was prepared from subcutaneous abscess tissue of rats for flow cytometry. The abscesses were removed immediately after the rats have been killed by cervical dislocation. The abscess was rinsed with PBS for 3 times and cut into pieces with sterile scissors. 2.5 mL 4 mg mL⁻¹ collagenase I, 2.5 mL 4 mg mL⁻¹ dispase II and 2.5 mL 5 mg mL⁻¹ elastase were added and incubated at 37 °C for 2 h, vortexing every 15 min. After addition of 1 mL of fetal bovine serum, tissue suspension was strained through 70 μm filter and centrifuged at 400 × g for 5 min. The cell suspension of each group was divided into 2 parts, and re-suspended in PBS (containing 5% FBS). The cells were blocked with rat serum in 4 °C for 30 min. The piezoNPs was labeled with CY5-PEG-SH before injection. Cells were stained with 1 μL antibody (anti-CD11b/c and anti-HIS48), respectively. Then Dil (5 μM) was added at 4 °C for 30 min in the dark. Samples were incubated with staining antibody for 1 h at 4 °C in the dark, and washed with PBS twice. Cells were resuspended in 1 mL of PBS immediately before flow cytometry.

For macrophage/neutrophil depletion assay, rats received intraperitoneal injection of CL (5 mg mL⁻¹,1.5 mL per rat) for macrophage depletion and neutrophil depletion was achieved with intraperitoneal injection of 150 μg anti-Ly6G at day −1 and day 0 (day of infection).

Cell preparation

Animals were exposure to carbon dioxide to euthanasia. The marrow in isolated tibias and femurs from 6-to-8-week-old Wistar rats (obtained from Chales River, Beijing, China) was rinsed with RPMI 1640 via a syringe. The mixture was passed through a 70 μm cell strainer and the supernatant was discarded by centrifugation at 400 × g for 10 min. Cells were rinsed and resuspended with RPMI 1640 medium containing 20% FBS and 20 μg mL⁻¹ GM-CSF (granulocyte-macrophage CSF). BMDMs were seeded on 12-well plates (2 × 10⁶ cells/well) and cultured overnight. On the next day, cells were washed three times with PBS, and 0.1 mg mL⁻¹ piezoNPs in RPMI 1640 was added and incubated with the cells for 1 h. After piezoNPs being completely washed, US (0.1 W cm⁻²) was applied for 10 s. The cells were scraped off and re-suspended with 100 μL PBS before injection. Cells with medium only served as Mϕ; cells primed with piezoNPs as piezoMϕ; piezoMϕ treated with US as activated piezoMϕ.

Quantitative real-time PCR and RNA sequencing

BMDMs were seeded on 6-well plates (5 × 10⁶ cells/well) and cultured overnight. On the next day, cells were washed three times with PBS. Then 0.1 mg mL⁻¹ piezoNPs in RPMI 1640 was added and incubated with the cells for 1 h. After piezoNPs being completely removed, US (0.1 W cm⁻²) was applied for 10 s. Subsequently, S. aureus in RPMI1640 medium without antibiotics (MOI = 100) was centrifugated onto the cells (600 × g) and incubated at 37 °C for 1 h. Cells with medium only served as Mϕ. Only bacterial infected cells were used in the medium as infected controls. After 1 h, cells were de-attached, and washed. RNAzol® RT RNA extraction reagent (RN190, MRC, USA) was used to isolate the total RNA of the cells in line with the manufacturer’s instructions. The RNA concentration and purity of the samples were measured by ultramicrospectrophotometer (Thermo Fisher Scientific, Nano-drop 2000). Subsequently, RNA was reverse-transcribed to complementary DNA (cDNA) using the SPARKscript II All-in-one RT SuperMix for qPCR reagent kit with gDNA Eraser (SparkJade, AG0305, China). Quantitative real-time PCR assays were performed with 2 × SYBR Green qPCR Mix (SparkJade, AH0101, China) and the Real-Time PCR System (Roche, LightCycler 96). Transcript levels were quantified employing primer-probe sets (Supplementary Table 1) that were designed using the online ProbeFinder Software (Roche).

RNA sequencing results were provided by LC-Bio Technology (Hang Zhou, China). All submitted samples passed quality control using the R package FastQC. Raw read counts were processed by TMM normalization followed by log₂ transformation. Genes expressed in fewer than three samples were removed from further analysis. Limma voom was used to identify differentially expressed genes between each mutant cohort and the WT cohort with a cutoff fold-change > 2 and adjusted p-value < 0.05. RNASeq data are available in the Gene Expression Omnibus database under accession no. PRJNA1080539.

Bacterial phagocytosis assay

3 × 10⁵ cells were incubated with 1 mL 0.1 mg mL⁻¹ CY5-piezoNPs at 37 °C for different times (1, 2, 4, and 8 h). After being washed with PBS, 3 × 10⁷ pHrodo Green conjugated S. aureus were added and incubated at 37 °C or 4 °C for 1 h as a negative control. US was applied to the experimental group (0.1 W cm⁻² for 10 s). The bacteria phagocytosis was analyzed by flow cytometry (BD, Accuri C6 Plus) and observed by fluorescence microscopy (TH4-200, OLYMPUS).

ACT treatment of abscess

Rats were immunosuppressed with three intraperitoneal doses of CY (100 mg kg⁻¹) 3 days before infection. A suspension of S. aureus in Luria-Bertani medium (1 × 10⁸ CFU mL⁻¹ 100 μL) was subcutaneously injected in the rats’ back. An obvious infected abscess had arisen subcutaneously after 24 h post infection. Afterward, the rats were administered with 0.1 mL PBS, 0.1 mL Mϕ suspension (5 × 10⁵ cells), 0.1 mL piezoMϕ suspension (5 × 10⁵ cells), and 0.1 mL activated piezoMϕ suspension (5 × 10⁵ cells). After 24 h, the rats were euthanized using carbon dioxide, and their skin was exposed to photograph the abscess.. The abscess was anatomically measured for size.

ACT treatment of sepsis

S. aureus ATCC 12598 (1 × 10⁹ CFU/mL in saline, 0.1 mL bacteria suspension per injection) was inoculated via i.v. route, and the infected mice were randomly divided into 4 groups (n = 8). Six hours after the infection, the mice were administered with 0.1 mL PBS, 0.1 mL Mϕ suspension (2 × 10⁶ cells per mouse), 0.1 mL piezoMϕ suspension (2 × 10⁶ cells per mouse), and 0.1 mL activated piezoMϕ suspension (2 × 10⁶ cells per mouse) through the tail vein. At 6, 24, and 480 h after infection, blood was collected and tested for WBCs and LYMs by a blood cell analyzer (IDEXX, ProCyte Dx). After 24 and 480 h, the animals were euthanized and their organs (heart, kidneys, liver, spleen, lung) and ascites were collected. The obtained tissues were homogenized and CFU was determined by serial dilution and plating-counting. All the mice were evaluated by BWs and survival.

ACT treatment of lung infection

Mice were subcutaneously injected with 150 mg/kg body weight of cyclophosphamide in 200 μL of PBS, four days and one day (100 mg/kg body weight of cyclophosphamide) before the infection. At day 0, P. aeruginosa (ATCC 27853, 5 × 10⁹ CFU per mouse) was inoculated into the lungs via nasal inhalation, and the infected mice were randomly divided into 4 groups (n = 9 per group). Four hours after the infection, a single dose of cells (2 × 10⁶ cells per mouse) in different treatment groups was given through i.v. injection. Twice rinses of 0.8 mL PBS were given to the lungs 24 h after infection, obtaining 1.2 mL in total. After a 5-min centrifugation at 8000 × g and 4 °C, the supernatant was gathered. The lungs were collected and fixed in 4% paraformaldehyde. After embedding in paraffin routinely, the tissues were cut into sections and stained with H&E. The P. aeruginosa was fluorescent pre-labeled by SYTO 9 before infection, and piezoMϕ by CellTacker before cell transfer. The lung tissue was removed and fixed with 4% PFA after 1 h. Paraffin embedding was followed by section. The samples were then incubated with anti-CD31 (dilution 1:2000, ab182981, abcam) primary antibodies overnight at 4 °C. The tissue samples were washed three times with immunostaining buffer and incubated with goat anti-rat CY3 antibodies for 1 h at 37 °C. Twenty-four hours after the inoculation, the animals were euthanized and their lungs were harvested and homogenized for CFU determination. Kinetics of biodistribution of the injected macrophages labelled with DIR lipid dyes was tracked by IVIS (IVIS Spectrum, PerkinElmer). At 1, 24, and 72 h of treatment, animals were exposure to carbon dioxide to euthanasia, main organs were removed, washed, and evaluated by IVIS Aura software.

Cell migration test

BMDMs was inoculated into the upper chamber of the 24 well plate transwell chamber (5 × 10⁵ cells/well), and cultured overnight with 1% FBS. The non-inflammation cells were separated into four groups and subjected to different treatments: NC (upper chamber, 5 × 10⁵ Mϕ), piezoMϕ (upper chamber, 5 × 10⁵ piezoMϕ), Activated piezoMϕ (upper chamber, 5 × 10⁵ activated piezoMϕ), PC (upper chamber, 5 × 10⁵ Mϕ). While 1% FBS medium was used in the other groups for a 24-h culture period, 20% FBS medium was used in the lower chamber of the PC group. In the inflammation group, S. aureus (MOI = 100) was added to the lower chamber and co-cultured for 1 h. The culture medium containing antibiotics was replaced in the lower chamber and continued for 24 h. The transwell chamber was fixed in 4% polyformaldehyde for 20 min and then washed with PBS three times. After dyeing with 0.1% crystal queviolet at room temperature for 20 min, then washed with PBS twice. The dried transwell chamber was imaged and analyzed through a microscope.

ACT treatment of MRSA peritonitis

Mice were immunosuppressed by cyclophosphamide as described above. At day 0, MRSA (ATCC 33591, 1 × 10⁸ CFU/mL in saline, 0.1 mL bacteria suspension per injection) was inoculated via the i.p. route, and the infected mice were randomly divided into 4 groups (n = 9). Two hours after the infection, a single dose of cells (2 × 10⁶ cells per mouse) in different treatment groups were given through i.v. injection. Twelve hours after the inoculation, the animals were euthanized and their organs (kidneys, liver, spleen, lung) and ascites were collected. To collect the ascites sample, 3 mL PBS was given through i.p. injection, and the abdomens of the mice were gently massaged. Peritoneal fluid was then removed from the peritoneum by syringe. The organs and tissue were homogenized and CFU was determined by serial dilution and plating.

Statistical analysis

All data were expressed in this manuscript as mean ± SD. All the results have been performed independently three times. For each test, P values <0.05 were considered statistically significant.

Data availability

All data are available in the main text or the supplementary materials. RNA-Seq have been deposited in the NCBI Sequence Read Archive under access code SRP492084. Source data files are provided with this paper. Source data are provided with this paper.

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Acknowledgements

Thanks Prof. Wanjun Chen and Prof. Wei Zhao for their useful discussion and suggestion. This work was supported by National Natural Science Foundation of China (82470981, 52100191, 82320108004, 2023YFC2506300), Taishan Scholars Program of Shandong Province (ts20190975 and tsqn201909180), Shandong Province Key Research and Development Program (2024CXPT090, 2021ZDSYS18, and 2022CXGC020511), Shandong Province Major Scientific and Technical Innovation Project (2021SFGC0502), Natural Science Foundation of Shandong Province (ZR2024YQ055), Independent Training and Innovation Team in Jinan (202228055), Qilu Young Scholar Foundation of Shandong University, National Clinical Key Specialty (Periodontology) Construction Project.

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Department of Biomaterials & Periodontology, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Research Center of Dental Materials and Oral Tissue Regeneration & Shandong Provincial Clinical Research Center for Oral Diseases, Jinan, China
Xiaoyi Liu, Wenxiu Xu, Junkun Feng, Ying Wang, Kai Li, Yi Chen, Wenjun Wang, Weiwei Zhao, Shaohua Ge & Jianhua Li

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Xiaoyi Liu
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Contributions

Conceptualization: J.L. Methodology: X.L., W.X., J.F., Y.W., K.L., Y.C., W.W., and W.Z. Investigation: X.L. Visualization: X.L. and J.L. Funding acquisition: J.L., S.G., W.W., and W.Z. Project administration: J.L. and S.G. Supervision: J.L. and S.G. Writing – original draft: X.L. and J.L. Writing – review & editing: X.L., J.L., and S.G.

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Liu, X., Xu, W., Feng, J. et al. Adoptive cell transfer of piezo-activated macrophage rescues immunosuppressed rodents from life-threating bacterial infections. Nat Commun 16, 1363 (2025). https://doi.org/10.1038/s41467-025-56460-2

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Received: 01 February 2024
Accepted: 14 January 2025
Published: 04 February 2025
DOI: https://doi.org/10.1038/s41467-025-56460-2