The potential role of non-classical monocytes in preventing malarial parasitemic recurrence in a mouse model

The potential role of non-classical monocytes in preventing malarial parasitemic recurrence in a mouse model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The potential role of non-classical monocytes in preventing malarial parasitemic recurrence in a mouse model Jiaqin Fang, Suilin Chen, Yuanli Gao, Yongling Fan, Shuai Guo, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5928249/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Frequent recurrence is responsible for persistent Plasmodium infection after the acute stage. Our previous study demonstrated that phagocytic cells are essential for controlling Plasmodium chabaudi chabaudi AS ( P. chabaudi ) recurrence. Nevertheless, the specific type of phagocytic cells involved in controlling P. chabaudi recurrence, as well as their underlying molecular mechanisms of action, remain elusive. Methods Single-cell RNA sequencing (scRNA-seq) was employed to analyze splenic phagocytic cells during both the acute and recurrent phases of P. chabaudi infection. The frequencies of red pulp macrophages (RPMs), classical monocytes (CMs), and non-classical monocytes (NCMs) were detected by flow cytometry. Low-dose clodronate liposomes (CLs) and CCR2 −/− mice were used to investigate the protective role and origin of NCMs. Results Using scRNA-seq, we found that NCMs declined during the acute stage of P. chabaudi blood-stage infection, and then expanded rapidly in the recurrence stage. The changing trend of NCMs was confirmed by flow cytometry. To explore the potential role of NCMs in controlling parasitemic recurrence, NCMs were reduced by a low-dose of CLs during the recurrence stage, which significantly elevated the P. chabaudi parasitemia. Additionally, no significant difference in the proportion of splenic NCMs or CMs within the monocyte population was observed between the infected CCR2 −/− mice and their control littermates, suggesting that the transition from CMs to NCMs may not occur in this model. Conclusions In summary, our results indicate that NCMs potentially play a protective role in preventing malarial parasitemic recurrence, offering valuable insights into immune-based interventions against Plasmodium infection and potentially contributing to the prevention of malaria transmission. Malaria parasite NCMs recurrence CCR2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Malaria remains a highly prevalent and deadly disease worldwide. In 2022, it was estimated that there were 249 million cases of malaria, leading to approximately 608 000 deaths. More than three-quarters of deaths occur in children under 5 years of age, with the majority occurring in Sub-Saharan Africa [ 1 ]. A highly efficacious vaccine could greatly mitigate the global malaria burden [ 2 , 3 ]. However, such a vaccine for controlling malarial infections remains elusive. Naturally acquired immunity against malaria gradually develops after years of continuous exposure to Plasmodium parasites [ 4 ]. Immunity to malaria is primarily linked to robust antibody response and the availability and function of helper CD4 + T cells and cytotoxic CD8 + T cells. These immune cells play pivotal roles in combating both the asymptomatic liver-stage and symptomatic blood-stage of Plasmodium infection [ 5 , 6 ]. Parasite-specific CD4 + T cells serve as the primary immune-protective agents, effectively controlling blood-stage malaria infections [ 5 ]. In acute blood-stage malarial infections, parasite-specific CD4 + T cells undergo differentiation, primarily into T helper (Th)1 and follicular helper (Tfh) cells [ 7 ]. CD4 + Th1 cells produce IFN-γ, which prompts macrophages to eliminate captured parasites [ 8 ]. Tfh cells provide help to B cells and are indispensable for germinal center formation, affinity maturation, and the generation of protective anti-parasitic antibodies [ 9 ]. However, Plasmodium parasites have devised intricate mechanisms to evade host immune clearance, including antigenic diversity and the induction of immune effector cell apoptosis, resulting in frequent recrudescence [ 10 , 11 ]. Thus, Plasmodium infection often transitions from acute to chronic, persists at low levels for extended periods, ranging from several months to years, and serves as a reservoir that sustains ongoing malaria transmission [ 12 – 14 ]. Therefore, understanding the immune response that controls malaria recurrence is important for blocking malaria transmission. Although parasite-specific CD4 + Th1 and Tfh cells are crucial in the host's immune response against acute blood-stage malaria infection, these immune cells tend to be exhausted during the chronic infection stage [ 15 , 16 ], which implies that the adaptive immune system may lose its ability to effectively control chronic infection. Recently, γδ T cells have been confirmed as essential in preventing the parasitemic recurrence via their secretion of M-CSF [ 17 ]. In our previous research, we employed the Plasmodium chabaudi chabaudi AS ( P. chabaudi ) infection model to explore the dynamic immune change between the acute and recurrent phases [ 18 ]. P. chabaudi infection exhibit frequent recurrence, analogous to the recrudescence observed in human malaria. By using the high-dose clodronate liposomes (CLs) treatment, our previous study found that phagocytic cells play a pivotal role in controlling the recurrence of P. chabaudi [ 18 ]. These findings underscore the significance of phagocytic cells in effectively controlling chronic malarial infection. Monocytes and macrophages are regarded as the main phagocytic effector cells that play a crucial role in the removal of invading pathogens. Monocytes are categorized into two primary subsets: classical monocytes (CMs) and non-classical monocytes (NCMs), distinguished by their distinctive expression patterns of specific surface molecules [ 19 ]. Although CMs, also known as inflammatory or Ly6C hi monocytes, have been proven to replenish splenic red pulp macrophages (RPMs) and are crucial in controlling acute-stage parasitemia [ 20 , 21 ], there are no reports exploring the role of NCMs in malaria infection. To precisely identify the roles of CMs, NCMs and RPMs in modulating malaria recurrence, we performed an in-depth analysis of our previously published single-cell RNA sequencing (scRNA-seq) data [ 18 ] and extracted the monocytes and RPMs for further analysis. After clustering analysis, phagocytes were classified into three populations based on the cell-specific molecular marker genes: RPMs ( Adgre1 , Spic , Vcam1 ), CMs ( Itgam , Csf1r , Ly6c2 , Ccr2 ), and NCMs ( Itgam , Csf1r , Cx3cr1 ). Our results revealed dynamic changes in monocytes and macrophages during the acute and parasitemic recurrence stages. Compared to CMs, RPMs and NCMs expanded rapidly during the recurrence stage. Reduction of NCMs with a low-dose of CLs during the recurrence stages significantly elevated the P. chabaudi parasitemia. Moreover, during both the acute and recurrent phases of P. chabaudi infection, CCR2 −/− infected mice exhibited no significant changes in the percentages of splenic NCMs (Live/Dead − Ly6G − CD11b + F4/80 int Ly6C − ) and CMs (Live/Dead − Ly6G − CD11b + F4/80 int Ly6C + ) within the monocyte population, when compared to their littermate controls. Thus, these findings suggest that the expansion of NCMs in spleen may not be directly attributed to the conversion of CMs during P. chabaudi infection. Taken together, our findings indicate that NCMs play a potential role in controlling parasitemic recurrence, which deepens our understanding of the determinants of immunity against Plasmodium infections. Methods Mice C57BL/6 mice were purchased from GemPharmatech (Nanjing, Jiangsu, China). CCR2 −/− mice (B6.129S4- Ccr2 tm1Ifc /J) were obtained from professor Li Tang (Laboratory of Hepatology and Immunology, State Key Laboratory of Proteomics, Beijing, China). All mice were bred and maintained in our pathogen-free animal facility. Only female mice, aged 6–8 weeks, were used in our study. All mouse procedures were approved by the Animal Ethics Committee of the Army Medical University (Third Military Medical University) Institute of Medical Research, under the approval number AMUWEC20230344. Parasite and infection Plasmodium chabaudi chabaudi AS ( P. chabaudi ) was maintained in our laboratory. For infection studies, female mice were challenged intraperitoneally (i.p.) with 1×10 6 P. chabaudi -parasitized red blood cells (pRBCs). Parasitemia was assessed by examining Giemsa-stained thin blood smears every other day following infection. scRNA-seq analysis The scRNA-seq data was sourced from our previously published research [ 18 ], and the raw data has been deposited in the Gene Expression Omnibus (GEO) database under the accession number GSE192930. To analyze these scRNA-seq data, we employed the Seurat package (version 4.4.0) within the R programming environment (version 4.3.2) to perform unsupervised clustering of individual cells. Specifically, the Uniform Manifold Approximation and Projection (UMAP) method was employed for dimensionality reduction. In our previously published article [ 18 ], we classified spleen cells from P. chabaudi -infected mice into eight distinct populations: B cells, dendritic cells (DCs), RPMs, monocytes, neutrophils, NK cells, platelets, and T cells. In the current study, we selected RPMs and monocytes for further re-clustering analysis. General cell types were annotated by examining the expression of cell type-specific marker genes, along with genes that contribute to the heterogeneity observed within clusters. The genes of Adgre1 , Spic , and Vcam1 were used to delineate RPMs; Itgam , Csf1r , Ly6c2 , and Ccr2 for CMs; and Itgam , Csf1r , and Cx3cr1 for NCMs. Additionally, utilizing the Seurat package in R (version 4.4.0), we conducted differential gene expression analysis to compare the expression profiles between the two groups. Flow cytometry analysis The following antibodies were used in our experiments for flow cytometry analysis: anti-mouse/human CD11b (PerCP/Cyanine5.5, clone M1/70, BioLegend), anti-mouse Ly6G (APC, clone 1A8, BioLegend), anti-mouse Ly6C (PE, clone HK1.4, BioLegend), anti-mouse F4/80 (FITC, clone BM8, BioLegend), anti-mouse CD11c (APC, 117310, clone N418, BioLegend). All above antibodies were used at a 1:100 dilution. LIVE/DEAD™ Fixable Violet Staining kit (Invitrogen) was used at a 1:250 dilution to exclude the dead cells. For flow cytometry staining, spleens were collected at the indicated days, and single-cell suspensions of splenocytes were obtained. For surface staining, 1×10 6 splenocytes were firstly incubated with anti-CD16/32 antibodies (clone 93, BioLegend) to block Fc receptors. Then the splenocytes were stained with the antibodies of interest for 45 min. The cells were acquired on a FACSCanto II instrument (BD Bioscience, SanJose, CA, United States) and FlowJo v10 software (Tree Star Inc.) was used to analyzed the data. CD11b, Ly6G, F4/80, Ly6C and CD11c were used to define the different cell populations, including the neutrophils, RPMs, CMs, NCMs, and DCs. Low-dose CLs-based treatment C57BL/6 mice were injected intravenous (i.v.) with a single dose of 6.5 mg/kg body weight of clodronate liposomes (CLs) or PBS-loaded control liposomes (PBSL) (Liposoma BV) in a final volume of 200µl when the parasitemia in acute stage declined almost to zero. The impact of liposomes was evaluated 24 hours post-injection through flow cytometry analysis of splenic phagocytic cells, including NCMs (Live/Dead − Ly6G − CD11b + F4/80 int Ly6C − ), CMs (Live/Dead − Ly6G − CD11b + F4/80 int Ly6C + ), RPMs (Live/Dead − Ly6G − CD11b − F4/80 + ), DCs (Live/Dead − CD11c hi ), and neutrophils (Live/Dead − CD11b + Ly6G + ). Immunofluorescence The spleens were surgically removed from C57BL/6 mice on day 0, 16 post-infection, with low-dose CLs or control PBSL treatment. Spleens were promptly snap-frozen and cryosectioned into 8-micron thick slices, which were then mounted onto glass slides. The sections were fixed with 4% paraformaldehyde in PBS for 30 minutes at room temperature (RT). Subsequently, they were washed three times with PBS and permeabilized with 0.1% saponin in PBS for 10 minutes at RT. The fixed sections were incubated overnight at 4 ℃ with primary antibodies diluted in a blocking solution, including anti-CD45R (B220) monoclonal antibody labeled with Alexa Fluor 488 (53-0452-82, Invitrogen, 1:25), and either anti-SIGN-R1 (05PB1435, BMA BIOMEDICALS, 1:400) or anti-MoMA-1 (11PB1616, BMA BIOMEDICALS, 1:400). After washing with PBS, the sections were incubated for 1 hour at RT with Streptavidin conjugated to Alexa Fluor™ 647 (S21374, Invitrogen, 1:100). Images were captured using the LSM 800 microscope system and analyzed with ZEN Imaging Software 3.4 (Zeiss). The Integrated Density (IntDen) of SIGN-R1 and MoMA-1 was quantified using ImageJ v2.1. Cells were stained with anti-CD45R (B220) for B cells, anti-SIGN-R1 for marginal zone macrophages (MZM) and anti-MoMA-1 for marginal metallophilic macrophages (MMM). Identification of CCR2 mice The offspring of the CCR2 −/− mice were identified by Polymerase Chain Reaction (PCR) and flow cytometry. For PCR analysis, the absence of CCR2 sequence were identified by using genomic DNA isolated from tail tissue. The primers set were FW1 (5’-CCACAGAATCAAAGGAAATGG-3’), RV1(5’-CACAGCATGAACAATAGCCAA G-3’), RV2 (5’-CCTTCTATCGCCTTCTTGAC G). For flow cytometry analysis, inflammatory monocytes (Ly6C hi monocytes, Ly6G − CD11b + Ly6C hi ) were identified to differentiate the CCR2 −/− mice from their wild-type littermates, as previously reported [ 21 ]. Statistical analysis Data are given as the means ± standard error (SD) unless otherwise stated. Statistical analysis was performed using GraphPad Prism version 8.0 (GraphPad Software, La Jolla, CA, United States). Statistically significant differences between the two groups were determined using the unpaired Student’s t-test, one-way ANOVA or two-way ANOVA. Statistical significance is indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, and 'ns' indicates 'not significant'. Results Single cell analysis reveals the dynamic change of monocytes and macrophages between acute and recurrence stages Our previous findings indicated that phagocytic cells participate in controlling parasitemic recurrence. To further investigate which type of phagocytic cells prevent this recurrence, scRNA-seq data from our previous study was further analyzed (Fig. 1 a) [ 18 ]. We extracted RPMs and monocytes and conducted re-clustering analysis. With a resolution of 0.8, the cells were divided into 13 clusters (Additional file 1: Fig. S1a). We used the genes Adgre1 (encoding F4/80), Spic (encoding Spi-C), Vcam1 (encoding VCAM1), Itgam (encoding CD11b), Csf1r (encoding CD115), Ly6c2 (encoding Ly6C), Ccr2 (encoding CCR2), and Cx3cr1 (encoding CX3CR1) to define the cell subpopulations. Spi-C is a characteristic transcription factor for RPMs, with F4/80 and VCAM1 typically exhibiting elevated expression levels on their surface [ 22 ]. Previous study has established that murine CMs are defined as CD11b + CD115 + Ly6C hi CCR2 hi CX3CR1 low , whereas NCMs are defined as CD11b + CD115 + Ly6C low CX3CR1 hi CCR2 low [ 23 ]. Therefore, we utilized the genes Itgam , Csf1r , Ly6c2 , Ccr2 , and Cx3cr1 to differentiate between CMs and NCMs. After conducting the clustering analysis, monocytes and macrophages were found to consist of three distinct subsets: CMs ( Itgam , Csf1r , Ly6c2 , Ccr2 ), NCMs ( Itgam , Csf1r , Cx3cr1 ), and RPMs ( Adgre1 , Spic, Vcam1 ). As shown in Fig. S1b, populations 3 and 8 correspond to CMs, population 6 corresponds to NCMs, and the populations 2, 4, 9, 10, 11 are classified as RPMs (Additional file 1: Fig. S1b; Fig. 1 b-d). The percentage of CMs substantially increased during the acute stage and subsequently decreased during the recurrent phase. However, a contrasting trend was observed in the percentages of NCMs and RPMs, with a decline during the acute stage and subsequent increase during the recurrent phase (Fig. 1 e). Since splenic RPMs are not crucial for controlling P. chabaudi infection [ 24 ], we postulated that NCMs, which markedly increased during the recurrence stage, potentially play a pivotal role in suppressing parasite recurrence. Plasmodium infection induces NCMs expansion in the transition of acute to recurrence stages To further explore the potential role of NCMs in controlling parasitemic recurrence, the changing trend in NCMs frequency and number was analyzed using flow cytometry. Specifically, splenocytes were collected from naive mice as well as mice infected with P. chabaudi at days 8, 12, and 16 post-infection for this analysis. Consistent with the scRNA-seq data, the proportion of NCMs (Live/Dead − Ly6G − CD11b + F4/80 int Ly6C − ) in either the splenocytes or monocytes continued to decline during the acute stage (day 8 post-infection), but increased rapidly during the recurrent phase (day 16 post-infection) (Fig. 2 a-c). Although the number of splenic NCMs on day 8 post-infection is higher than that of naïve mice, which contrasts with the trend observed in NCMs frequency, the number of NCMs remains significantly elevated during the recurrence stage (days 12 and 16 post-infection) compared to days 0 and 8 post-infection (Fig. 2 d). These results further suggest that NCMs may play a potential role in controlling parasitemic recurrence. NCMs, but not other phagocytes, were selectively reduced after low-dose CLs treatment in P. chabaudi recurrence To investigate the role of NCMs in controlling recurrent infections of P. chabaudi , it would be ideal to use conditional knockout mice that allow for inducible depletion of NCMs. However, such mice are not commercially available. Alternatively, the small-molecule inhibitor AZD8797 has been used to selectively reduce the population of Ly6C low NCMs [ 25 ]. However, we found that the administration of AZD8797 did not significantly deplete NCMs during the recurrent phase of P. chabaudi infection (data not shown). Therefore, alternative approaches are needed to specifically target and deplete NCMs. CLs are commonly used to specifically deplete phagocytes, including macrophages, monocytes, and DCs, in various organs and tissues [ 18 , 26 ]. However, the effectiveness of this depletion varies with the dose of CLs, with low doses demonstrating a more selective impact on specific phagocyte subsets. Therefore, we investigated whether the administration of low-dose CLs could reduce NCMs during the recurrent phase of P. chabaudi infection. Subsequently, we examined the frequencies and numbers of major phagocytic cell populations in spleen 24 hours after administering the low-dose CLs. These populations included RPMs (Live/Dead − Ly6G − CD11b − F4/80 + ), CMs (Live/Dead − Ly6G − CD11b + F4/80 int Ly6C + ), NCMs (Live/Dead − Ly6G − CD11b + F4/80 int Ly6C − ), DCs (Live/Dead − CD11c hi ) and neutrophils (Live/Dead − CD11b + Ly6G + ). As anticipated, intravenous administration of low-dose CLs on day 14 post-infection did not significantly affect the frequency and number of RPMs, CMs, DCs or neutrophils, but significantly reduced the frequency and number of splenic NCMs in infected mice (Fig. 3 a-c). Given that previous studies indicated low-dose CLs specifically target the depletion of MZM and MMM [ 27 ], we assessed both splenic MZM and MMM in parasite-infected mice treated with or without low-dose CLs using immunofluorescence assays. Remarkably, both MZM and MMM were absent in the infected mice, regardless of whether they were treated with low-dose CLs (Fig. 3 d, e). The depletion of MZM and MMM in the spleens of P. chabaudi -infected mice aligns with previous findings [ 28 ]. Thus, our data suggest that low-dose CLs can effectively reduce the total number of NCMs during the recurrent phase of P. chabaudi infection. Low-dose CLs treatment after acute parasitemia results in P. chabaudi recurrence Based on the aforementioned findings, we employed a dosage of 6.5 mg/kg of CLs to investigate the effect of NCMs reduction on parasitemic recurrence of P. chabaudi infection. Remarkably, intravenous administration of a low-dose of CLs in the recurrent phase (day 14 post-infection) significantly elevated the parasitemia levels, which were much higher than the peak in the control group (Fig. 4 a). However, no significant effect on the survival rate was observed after the administration of low-dose CLs in the recurrence stage (Fig. 4 b). Thus, our data strongly suggests a potential role for NCMs in preventing parasitemic recurrence. The transition of CMs was not essential for the development of NCMs during the P. chabaudi recurrence It has been observed that CMs from the blood continuously replenish the monocyte pool in the spleen and also differentiate into NCMs [ 29 ]. Given this, we wondered whether the fluctuation in the frequency of spleen NCMs following P. chabaudi infection is associated with the conversion of CMs. Since CCR2 is pivotal in facilitating the egression of CMs from the bone marrow into the bloodstream, mice deficient in CCR2 exhibit a deficiency in the majority of CMs [ 30 ]. Therefore, we used CCR2-deficient mice to test our hypothesis. Firstly, we referred to previous study and utilized inflammatory monocyte (Live/Dead − Ly6G − CD11b + Ly6C hi ) detection to validate the CCR2 −/− mice [ 21 ]. Consistent with previous finding, the percentage of splenic inflammatory monocytes in uninfected CCR2 −/− mice was significantly lower than that in uninfected littermate controls (Additional file 2: Fig. S2b, c). Notably, during P. chabaudi infection, there were no significant differences in the percentages of CMs and NCMs in the splenic monocytes between CCR2 −/− mice and their littermate controls (Fig. 5 a). Moreover, on days 0 and 7 post-infection, CCR2 −/− mice exhibited a significantly reduced number of splenic CMs compared to littermate controls. While the number of NCMs in uninfected CCR2 −/− mice was significantly higher than in their littermate counterparts, it was significantly lower than that of littermate controls on day 7 post-infection (Fig. 5 b). However, during the recurrent phase of P. chabaudi infection, no significant differences were observed between CCR2 −/− mice and littermate controls in terms of either the frequency or the number of splenic NCMs and CMs. Thus, these findings suggest that the increasing frequency and number of NCMs during the recurrent phase of P. chabaudi infection may not be attributable to the transition of CMs. Discussion Preventing the parasitemic recurrence is crucial in blocking malaria transmission [ 13 , 14 ]. However, our understanding of how immune cells control parasitic recurrence remains limited. Recent research has found that NCMs significantly expand in chronic rodent and human plasmodium infections [ 31 ]; however, the underlying biological significance of this phenomenon remains elusive. In the current study, our findings revealed that the frequency of NCMs significantly decreased in the acute stage but underwent a rapid increase during the recurrent phase of P. chabaudi infection. Notably, the reduction of NCMs during the recurrent phase led to a rapid increase in parasitemia, indicating their potential role in controlling parasitemic recurrence. Furthermore, using CCR2-deficient mice, we found that the increased NCMs frequency and number during P. chabaudi recurrence is not due to the conversion of CMs. These findings may highlight the specialized protective role of NCMs in preventing post-acute parasitemic recurrence, enhance our understanding of the host's ability to control chronic malaria infection, and provide theoretical support for blocking malaria transmission. Our study still has several limitations. Despite the proven efficacy of low-dose CLs in reducing NCMs, this approach remains inconclusive in establishing the definitive protective role of NCMs in preventing malaria recurrence. Previous studies have shown that other phagocytes, particularly neutrophils, contribute to protection against Plasmodium infection via antibody-dependent respiratory bursts [ 32 , 33 ]. However, our findings indicate that intravenous administration of low-dose CLs has no impact on the splenic neutrophil frequency and number. Notably, ablation of the Nr4a1 super-enhancer subdomain, referred to as E2, can effectively deplete NCMs [ 34 ]. However, we are still devoid of strategies to generate conditional knockout mice that selectively deplete NCMs during the recurrent phase of P. chabaudi infection. Recent studies have highlighted the high-affinity Cx3cr1 small-molecule inhibitor, AZD8797, in selectively diminishing the population of Ly6C low NCMs [ 25 ]. Consequently, we have adopted the method described in this literature, utilizing the Cx3cr1 inhibitor, AZD8797, to specifically deplete NCMs, aiming to clearly elucidate the pivotal role of NCMs in regulating the recurrent phase of P. chabaudi infection. Nevertheless, our preliminary experiments revealed that the frequency and number of NCMs remained unaffected following the administration of AZD8797 during the recurrent phase of P. chabaudi infection (data not shown). Previous study has indicated that intermediate monocytes may play a crucial role in controlling Plasmodium infections by phagocytosing and clearing infected erythrocytes [ 35 ]. Therefore, further research is essential to conclusively clarify the protective roles of NCMs and intermediate monocytes in preventing malaria recurrence. Meanwhile, the effective mechanism underlying the suppression of parasitemic recurrence by NCMs remain unclear. Previous research has predominantly centered on the function of NCMs in clearing debris through Fcγ-mediated phagocytosis in vessels and monitoring endothelial cell integrity. Furthermore, neutrophils in peripheral blood, rather than monocytes, can mediate phagocytosis of sporozoites through Fcγ-receptor (FcγR). However, it remains unclear whether different subsets of monocytes in the spleen can exert immune-protective effects through FcγR [ 36 ]. Therefore, examining the expression of FcγR on various subsets of splenic monocytes will aid in understanding their roles in controlling Plasmodium infection. Additionally, a recent study revealed that NCMs robustly express the immune checkpoint molecule PD-L1 (CD274) [ 37 ]. Notably, under inflammatory conditions, PD-L1 + NCMs exhibit immunomodulatory functions, promoting T cell apoptosis in tertiary lymphoid organs (TLOs) [ 37 ]. Moreover, NCMs exhibit heightened production of inflammatory cytokines in response to viral and nucleic acid stimuli, mediated by TLR7 [ 38 , 39 ]. They also trigger the recruitment and activation of innate immune cells, including NK cells and neutrophils, through TNF-α-mediated upregulation of E-selectin on endothelial cells [ 19 , 40 ]. Considering the intricate role of NCMs in the immune response, we still lack clues regarding their protective effects against malaria recurrence. Previous studies have demonstrated that monocytes can exert immune protection against blood-stage malaria infection by phagocytosing infected erythrocytes. Additionally, monocytes may also contribute to immune defense through antibody-dependent cellular inhibition [ 41 , 42 ]. However, this study has not yet definitively established whether NCMs also possess such protective effects. Therefore, further research is necessary to clarify the protective mechanisms of NCMs during the recurrent phase of P. chabaudi infection. CMs derive from myeloid-committed progenitors residing in the bone marrow (BM) and subsequently traverse into the circulation via a CCR2-dependent mechanism [ 30 , 43 ]. Conversely, the precise origin of NCMs continues to be enigmatic. Recent studies have revealed that NCMs can be derived from CM transitions [ 29 , 19 ]. CMs and NCMs have biological interconnections and show minimal differences in chromatin organization [ 44 ]. However, our findings demonstrate that the percentage of splenic NCMs and CMs within the monocyte population exhibited no significant differences between CCR2 −/− mice and their control littermates during the P. chabaudi infection, partially aligning with prior research findings [ 29 ]. These findings suggest that the transition to NCMs occurs independently of CCR2, a molecule exclusively expressed by CMs, in the context of P. chabaudi infection. The underlying mechanism may be attributed to trapped CMs in the BM of CCR2-deficient mice, resulting in a marked depletion of CMs [ 30 ]. Additionally, the transformation of CMs into NCMs appears to be less efficient in aged mice [ 37 ]. These observations suggest that the origin of NCMs is complex. Consistent with prior report, our findings also reveal a significant increase in the number of CMs in the spleen of CCR2 −/− mice during the later stages of P. chabaudi infection [ 21 ]. These observations imply that, apart from CMs, other immune cells may also contribute to replenishing splenic CMs reservoir during chronic P. chabaudi infection. Furthermore, our research revealed that the number of NCMs in the spleens of CCR2 −/− uninfected mice was significantly higher than in their control littermates. However, the underlying molecular mechanisms driving this phenomenon remain elusive. Conclusions The significance of NCMs in both health and disease contexts is increasingly recognized; nonetheless, their specific contribution to malarial infection remains undefined. In the present study, we demonstrated that NCMs, which expanded during the recurrent phase of P. chabaudi infection, have the potential role in controlling chronic malaria infection. Notably, this expansion may not originate from the transition of CMs. Therefore, our findings not only contribute to understanding how innate immune cells control the recrudescence of plasmodium infection, but also lay a theoretical foundation for the prevention of malaria transmission. Abbreviations scRNA-seq single-cell RNA sequencing RPMs red pulp macrophages CMs classical monocytes NCMs non-classical monocytes CLs clodronate liposomes PBSL PBS-loaded control liposomes MZM marginal zone macrophages MMM marginal metallophilic macrophages P. chabaudi Plasmodium chabaudi chabaudi AS i.p. intraperitoneally pRBCs parasitized red blood cells UMAP Uniform Manifold Approximation and Projection DCs dendritic cells Th1 T helper1 cells Tfh T follicular helper cells IntDen Integrated Density TLOs tertiary lymphoid organs BM bone marrow. Declarations Acknowledgements We thank Jiayin Biotechnology Ltd. (Shanghai, China), for the assistance with scRNA assay. We would like to thank Editage (www.editage.cn) for English language editing. Funding This work was supported by the National Natural Science Foundation of China (No. 82172296), the Natural Science Foundation of Chongqing (No. CSTB2023NSCQ-MSX0788). Availability of data and materials The data supporting the conclusions of this article are included within the article. Data of scRNA used in this study are publicly available from the GEO database at www.ncbi.nlm.nih.gov/geo/ under accession number GSE192930. Author contributions TL and WX conceived the experiment and wrote the paper. JF, SC, YG, YF, SG, XL, HL and JZ performed the research and analyzed the data. All authors read and approved the final manuscript. Ethics approval and consent to participate All animal studies were reviewed and approved by the Animal Ethics Committee of the Third Military Medical University Institute of Medical Research, Chongqing, China (ethical approval number: AMUWEC20230344). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details 1 Department of Pathogenic Biology, Army Medical University (Third Military Medical University), Chongqing, China. 2 Clinical Laboratory Diagnostic Center, General Hospital of Xinjiang Military Region, Urumqi, China. 3 Institute of Immunology, Army Medical University (Third Military Medical University), Chongqing, China. 4 Key Laboratory of Extreme Environmental Medicine, Ministry of Education of China, Chongqing, China. References World Health Organization. World Malaria Report 2022. WHO Global Malaria Programme. https://www.who.int/teams/global-malaria-programme/reports/ world-malaria-report-2022 El-Moamly AA, El-Sweify MA. Malaria vaccines: the 60-year journey of hope and final success-lessons learned and future prospects. Trop Med Health. 2023;51:29. https://doi.org/10.1186/s41182-023-00516-w . Datoo MS, Dicko A, Tinto H, Ouédraogo JB, Hamaluba M, Olotu A, et al. 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J Clin Invest. 2007;117:902–9. https://doi.org/10.1172/JCI29919 . Mildner A, Schönheit J, Giladi A, David E, Lara-Astiaso D, Lorenzo-Vivas E, et al. Genomic characterization of murine monocytes reveals C/EBPbeta transcription factor dependence of Ly6C(-) cells. Immunity. 2017;46:849–62. https://doi.org/10.1016/j.immuni.2017.04.018 . Additional Declarations No competing interests reported. Supplementary Files floatimage6.jpeg Additional file 1: Fig. S1. Definition of cell clusters. (a) Cell clustering performed at a resolution of 0.8. (b) Violin plots illustrating the expression levels of key marker genes across each cell cluster. floatimage7.jpeg Additional file 2: Fig. S2. Identification of CCR2 -/- mice by PCR and FACS analysis. (a) A PCR-based method for genotyping mice that have been knocked out for the CCR2 gene. (b) Gating strategy of splenic inflammatory monocytes (LY6C hi monocytes, Live/Dead - Ly6G - CD11b + Ly6C hi ). (c) The percentage of the inflammatory monocytes from the spleen of the uninfected CCR2 -/- mice and control littermates. n = 3 for each group. The data represent the mean ± SD. Unpaired Student’s t-test was used for statistical analysis. The experiments were repeated three times. *** p < 0.001. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5928249","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":410234313,"identity":"c4b6608c-3244-4344-b07b-cf1762c70394","order_by":0,"name":"Jiaqin Fang","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiaqin","middleName":"","lastName":"Fang","suffix":""},{"id":410234314,"identity":"b34a1c52-63ef-4cbb-ac82-501ecd988c2b","order_by":1,"name":"Suilin Chen","email":"","orcid":"","institution":"General Hospital of Xinjiang Military Region,","correspondingAuthor":false,"prefix":"","firstName":"Suilin","middleName":"","lastName":"Chen","suffix":""},{"id":410234315,"identity":"5b1fd1bf-a331-4631-9d27-1a65683ab857","order_by":2,"name":"Yuanli Gao","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuanli","middleName":"","lastName":"Gao","suffix":""},{"id":410234316,"identity":"df862f8f-2c96-4e5d-af65-473faf5e0dc7","order_by":3,"name":"Yongling Fan","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yongling","middleName":"","lastName":"Fan","suffix":""},{"id":410234317,"identity":"e725a3df-c86d-410e-b74e-fed0b4d55e06","order_by":4,"name":"Shuai Guo","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shuai","middleName":"","lastName":"Guo","suffix":""},{"id":410234318,"identity":"3170fc0a-1fc7-4cab-864b-502e2d620061","order_by":5,"name":"Xiuxiu Li","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiuxiu","middleName":"","lastName":"Li","suffix":""},{"id":410234319,"identity":"b3cf5eb1-8bd0-47e7-b9d1-5276afb74928","order_by":6,"name":"Hangyu Li","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hangyu","middleName":"","lastName":"Li","suffix":""},{"id":410234320,"identity":"aa714edf-aed2-4c70-886c-3926bea408ae","order_by":7,"name":"Jian Zhou","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Zhou","suffix":""},{"id":410234321,"identity":"4564acbc-c024-4499-9660-fe793f61e46d","order_by":8,"name":"Wenyue Xu","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenyue","middleName":"","lastName":"Xu","suffix":""},{"id":410234322,"identity":"c49806ab-dbea-46ab-8498-bd9136579cf7","order_by":9,"name":"Taiping Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYBACPmYQacDAw8DAfAAqloBfCxszA2MDRAtbYgNxWhhAWsCAx5BILew85g9+FNyRMedf8/3RzZzDDPzsOQYMP3fgcxiPYWOPwTMeyxlvNzbnbjvMINnzxoCx9wx+LQ08Bod5DG6chWgxuJFjwMzYRsCWP2AtZx6CtdgTo6UZbMv5HkaILRIEtbAVzpYB+sXgBpvh7Nxt6TwSZ54VHOzFo4Wf//CGj2/+3LE3OH/4wefcbdZy/O3JGx/8xKMFCg4wMEgkgFk8UC4xWviJUTcKRsEoGAUjEgAAc0NQm1CiKyYAAAAASUVORK5CYII=","orcid":"","institution":"Army Medical University","correspondingAuthor":true,"prefix":"","firstName":"Taiping","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-01-30 07:08:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5928249/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5928249/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75356826,"identity":"7192553c-08d6-4071-b82a-e0a8543f5bc4","added_by":"auto","created_at":"2025-02-03 17:08:09","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2117084,"visible":true,"origin":"","legend":"\u003cp\u003escRNA-seq analysis revealed the dynamic changes of phagocytic cells during the \u003cem\u003eP. chabaudi\u003c/em\u003e infection. (\u003cstrong\u003ea\u003c/strong\u003e) Schematic representation of the experimental workflow. Splenocytes from five mice were isolated on days 0, 8, and 16 post-infection, and then pooled for 10×Genomics scRNA-seq analysis. The arrow highlights the specific day on which the mouse spleens were collected. (\u003cstrong\u003eb\u003c/strong\u003e) Unsupervised clustering was conducted on the UMAP to discern clusters of phagocytic cells exhibiting similar gene expression patterns. (\u003cstrong\u003ec\u003c/strong\u003e) UMAP visualization depicting the expression of canonical immune cell markers across each cell cluster. (\u003cstrong\u003ed\u003c/strong\u003e) Violin plots show the expression levels of representative marker genes within each cell cluster. (\u003cstrong\u003ee\u003c/strong\u003e) The relative fractions of immune cell populations among all splenocytes during \u003cem\u003eP. chabaudi\u003c/em\u003e infection.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5928249/v1/df61c5f1c6cabb8c7492d964.jpeg"},{"id":75356523,"identity":"f1fc58df-874b-43d7-828b-9858c35eed86","added_by":"auto","created_at":"2025-02-03 17:00:09","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":271058,"visible":true,"origin":"","legend":"\u003cp\u003eFACS verified the expansion of NCMs in the recurrence stages of \u003cem\u003eP. chabaudi\u003c/em\u003e infection. (\u003cstrong\u003ea\u003c/strong\u003e) The gating strategy of NCMs (Live/Dead\u003csup\u003e-\u003c/sup\u003eLy6G\u003csup\u003e-\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003eint\u003c/sup\u003eLy6C\u003csup\u003e-\u003c/sup\u003e) and CMs (Live/Dead\u003csup\u003e-\u003c/sup\u003eLy6G\u003csup\u003e-\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003eint\u003c/sup\u003eLy6C\u003csup\u003e+\u003c/sup\u003e) subsets. (\u003cstrong\u003eb\u003c/strong\u003e) Statistical analysis of the percentages of NCMs in the spleen from the infected mice at the indicated time points (n = 4 for each time point). (\u003cstrong\u003ec\u003c/strong\u003e) Statistical analysis of the percentages of NCMs and CMs in the splenic monocytes from the infected mice at the indicated time points (n = 4 for each time point). (\u003cstrong\u003ed\u003c/strong\u003e) Comparison of number of NCMs at the indicated time point (n=4 for each time point). The data represent the mean ± SD. One-way ANOVA was used for statistical analysis. The data are representative of at least two independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5928249/v1/a993dd9870a1fa74b08bfac4.jpeg"},{"id":75356827,"identity":"e3559eef-7bec-4cee-8703-c0c48fb28639","added_by":"auto","created_at":"2025-02-03 17:08:09","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":271524,"visible":true,"origin":"","legend":"\u003cp\u003eLow-dose CLs treatment resulted in a significant reduction of NCMs. (\u003cstrong\u003ea\u003c/strong\u003e) Gating strategy of neutrophils (Live/Dead\u003csup\u003e-\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e), DCs (Live/Dead\u003csup\u003e-\u003c/sup\u003eCD11c\u003csup\u003ehi\u003c/sup\u003e), RPMs (Live/Dead\u003csup\u003e-\u003c/sup\u003eLy6G\u003csup\u003e-\u003c/sup\u003eCD11b\u003csup\u003e-\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e), NCMs (Live/Dead\u003csup\u003e-\u003c/sup\u003eLy6G\u003csup\u003e-\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003eint\u003c/sup\u003eLy6C\u003csup\u003e-\u003c/sup\u003e) and CMs (Live/Dead\u003csup\u003e-\u003c/sup\u003eLy6G\u003csup\u003e-\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003eint\u003c/sup\u003eLy6C\u003csup\u003e+\u003c/sup\u003e) subsets in infected mice are shown. Statistical analysis of the percentage (\u003cstrong\u003eb\u003c/strong\u003e) and number (\u003cstrong\u003ec\u003c/strong\u003e) of NCMs, CMs, RPMs, DCs and neutrophils in spleen of infected mice after low-dose CLs treatment (n = 4 for each group). (\u003cstrong\u003ed\u003c/strong\u003e) Immunofluorescence analysis showing MZM and MMM absence during the recurrence stage of infected mice. Cells were stained with anti-CD45R (B220) for B cells (green), anti-SIGN-R1 for MZM (red, above) and anti-MoMA-1 for MMM (red, below). Scale bars: 100 μm. (\u003cstrong\u003ee\u003c/strong\u003e) Quantitative analysis of the MZM and MMM by measuring the Integrated Density (IntDen) of SIGN-R1 and MoMA-1 respectively (n = 3 per group). \u0026nbsp;The data represent the mean ± SD. The unpaired Student’s t-test (\u003cstrong\u003eb\u003c/strong\u003e and \u003cstrong\u003ec\u003c/strong\u003e) or one-way ANOVA (\u003cstrong\u003ee\u003c/strong\u003e) were used for statistical analysis. The data are representative of at least three independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ns, not statistically significant.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5928249/v1/e39ebdf4dca4542939bcc020.jpeg"},{"id":75356828,"identity":"e83b06c3-3e38-44df-aa5d-b945912a92d3","added_by":"auto","created_at":"2025-02-03 17:08:09","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":107346,"visible":true,"origin":"","legend":"\u003cp\u003eLow-dose CLs treatment results in\u003cem\u003e P. chabaudi \u003c/em\u003erecurrence. To assess for the effect of NCMs reduction in \u003cem\u003eP. chabaudi\u003c/em\u003e recurrence, C57BL/6 mice were challenged i.p. with 1×10\u003csup\u003e6\u003c/sup\u003e pRBCs and administrated with low-dose CLs or control PBSL when the parasitemia declined almost to zero (day14 post-infection). The outcome of infection was monitored by assessing parasitemia (\u003cstrong\u003ea\u003c/strong\u003e) and survival rate (\u003cstrong\u003eb\u003c/strong\u003e). n = 5 for each group. The data represent the mean ± SD. Two-way ANOVA was used for statistical analysis. \u0026nbsp;The data are representative of at least three independent experiments. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ns, not statistically significant.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5928249/v1/78352ffcc7a3c29f29e6c013.jpeg"},{"id":75356526,"identity":"03892f11-57fb-40b1-bb0e-444dc33c7565","added_by":"auto","created_at":"2025-02-03 17:00:09","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":171004,"visible":true,"origin":"","legend":"\u003cp\u003eExpansion of NCMs during the \u003cem\u003eP. chabaudi\u003c/em\u003e recurrent phase was not originated from CMs.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Percentage of CMs and NCMs in the monocytes of WT and CCR2\u003csup\u003e-/-\u003c/sup\u003e mice on days 0, 7, 11, 16 and 20 post-infection were shown (n = 3 for each group ). (\u003cstrong\u003eb\u003c/strong\u003e) Statistical analysis of the number of CMs and NCMs in the spleen from the infected mice at the indicated time points (n = 3 for each time point). The data represent the mean ± SD. Two-way ANOVA was used for statistical analysis. The data are representative of at least two independent experiments. WT, wild type; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ns, not statistically significant.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5928249/v1/07e21f779ce027e605c4d298.jpeg"},{"id":76188210,"identity":"8b807e8c-d8a1-468a-8fdd-71eb385273a3","added_by":"auto","created_at":"2025-02-13 09:02:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3205907,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5928249/v1/1008afa4-58bf-4dd7-883e-acbdd980107f.pdf"},{"id":75356518,"identity":"a2bd5557-2529-433a-b287-0f0a38cc30d6","added_by":"auto","created_at":"2025-02-03 17:00:09","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":147215,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1: Fig. S1.\u003c/strong\u003e Definition of cell clusters. (\u003cstrong\u003ea\u003c/strong\u003e) Cell clustering performed at a resolution of 0.8. (\u003cstrong\u003eb\u003c/strong\u003e) Violin plots illustrating the expression levels of key marker genes across each cell cluster.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5928249/v1/a3e91b402e99064c8bd1252d.jpeg"},{"id":75356521,"identity":"f34c3df2-d3de-4433-b8a2-c6b3445f2683","added_by":"auto","created_at":"2025-02-03 17:00:09","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":149616,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 2: Fig. S2.\u003c/strong\u003e Identification of CCR2\u003csup\u003e-/-\u003c/sup\u003e mice by PCR and FACS analysis. (\u003cstrong\u003ea\u003c/strong\u003e) A PCR-based method for genotyping mice that have been knocked out for the CCR2 gene. (\u003cstrong\u003eb\u003c/strong\u003e) Gating strategy of splenic inflammatory monocytes (LY6C\u003csup\u003ehi\u003c/sup\u003e monocytes, Live/Dead\u003csup\u003e-\u003c/sup\u003eLy6G\u003csup\u003e-\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehi\u003c/sup\u003e). (\u003cstrong\u003ec\u003c/strong\u003e) The percentage of the inflammatory monocytes from the spleen of the uninfected CCR2\u003csup\u003e-/-\u003c/sup\u003e mice and control littermates. n = 3 for each group. The data represent the mean ± SD. Unpaired Student’s t-test was used for statistical analysis. The experiments were repeated three times. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5928249/v1/c0a98c67dc7b6b610e363fa2.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"The potential role of non-classical monocytes in preventing malarial parasitemic recurrence in a mouse model","fulltext":[{"header":"Background","content":"\u003cp\u003eMalaria remains a highly prevalent and deadly disease worldwide. In 2022, it was estimated that there were 249\u0026nbsp;million cases of malaria, leading to approximately 608 000 deaths. More than three-quarters of deaths occur in children under 5 years of age, with the majority occurring in Sub-Saharan Africa [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. A highly efficacious vaccine could greatly mitigate the global malaria burden [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, such a vaccine for controlling malarial infections remains elusive.\u003c/p\u003e \u003cp\u003eNaturally acquired immunity against malaria gradually develops after years of continuous exposure to \u003cem\u003ePlasmodium\u003c/em\u003e parasites [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Immunity to malaria is primarily linked to robust antibody response and the availability and function of helper CD4\u003csup\u003e+\u003c/sup\u003e T cells and cytotoxic CD8\u003csup\u003e+\u003c/sup\u003e T cells. These immune cells play pivotal roles in combating both the asymptomatic liver-stage and symptomatic blood-stage of \u003cem\u003ePlasmodium\u003c/em\u003e infection [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Parasite-specific CD4\u003csup\u003e+\u003c/sup\u003e T cells serve as the primary immune-protective agents, effectively controlling blood-stage malaria infections [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In acute blood-stage malarial infections, parasite-specific CD4\u003csup\u003e+\u003c/sup\u003e T cells undergo differentiation, primarily into T helper (Th)1 and follicular helper (Tfh) cells [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. CD4\u003csup\u003e+\u003c/sup\u003e Th1 cells produce IFN-γ, which prompts macrophages to eliminate captured parasites [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Tfh cells provide help to B cells and are indispensable for germinal center formation, affinity maturation, and the generation of protective anti-parasitic antibodies [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, \u003cem\u003ePlasmodium\u003c/em\u003e parasites have devised intricate mechanisms to evade host immune clearance, including antigenic diversity and the induction of immune effector cell apoptosis, resulting in frequent recrudescence [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Thus, \u003cem\u003ePlasmodium\u003c/em\u003e infection often transitions from acute to chronic, persists at low levels for extended periods, ranging from several months to years, and serves as a reservoir that sustains ongoing malaria transmission [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, understanding the immune response that controls malaria recurrence is important for blocking malaria transmission.\u003c/p\u003e \u003cp\u003eAlthough parasite-specific CD4\u003csup\u003e+\u003c/sup\u003e Th1 and Tfh cells are crucial in the host's immune response against acute blood-stage malaria infection, these immune cells tend to be exhausted during the chronic infection stage [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], which implies that the adaptive immune system may lose its ability to effectively control chronic infection. Recently, γδ T cells have been confirmed as essential in preventing the parasitemic recurrence via their secretion of M-CSF [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In our previous research, we employed the \u003cem\u003ePlasmodium chabaudi chabaudi AS\u003c/em\u003e (\u003cem\u003eP. chabaudi\u003c/em\u003e) infection model to explore the dynamic immune change between the acute and recurrent phases [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. \u003cem\u003eP. chabaudi\u003c/em\u003e infection exhibit frequent recurrence, analogous to the recrudescence observed in human malaria. By using the high-dose clodronate liposomes (CLs) treatment, our previous study found that phagocytic cells play a pivotal role in controlling the recurrence of \u003cem\u003eP. chabaudi\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These findings underscore the significance of phagocytic cells in effectively controlling chronic malarial infection.\u003c/p\u003e \u003cp\u003eMonocytes and macrophages are regarded as the main phagocytic effector cells that play a crucial role in the removal of invading pathogens. Monocytes are categorized into two primary subsets: classical monocytes (CMs) and non-classical monocytes (NCMs), distinguished by their distinctive expression patterns of specific surface molecules [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Although CMs, also known as inflammatory or Ly6C\u003csup\u003ehi\u003c/sup\u003e monocytes, have been proven to replenish splenic red pulp macrophages (RPMs) and are crucial in controlling acute-stage parasitemia [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], there are no reports exploring the role of NCMs in malaria infection. To precisely identify the roles of CMs, NCMs and RPMs in modulating malaria recurrence, we performed an in-depth analysis of our previously published single-cell RNA sequencing (scRNA-seq) data [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and extracted the monocytes and RPMs for further analysis. After clustering analysis, phagocytes were classified into three populations based on the cell-specific molecular marker genes: RPMs (\u003cem\u003eAdgre1\u003c/em\u003e, \u003cem\u003eSpic\u003c/em\u003e, \u003cem\u003eVcam1\u003c/em\u003e), CMs (\u003cem\u003eItgam\u003c/em\u003e, \u003cem\u003eCsf1r\u003c/em\u003e, \u003cem\u003eLy6c2\u003c/em\u003e, \u003cem\u003eCcr2\u003c/em\u003e), and NCMs (\u003cem\u003eItgam\u003c/em\u003e, \u003cem\u003eCsf1r\u003c/em\u003e, \u003cem\u003eCx3cr1\u003c/em\u003e). Our results revealed dynamic changes in monocytes and macrophages during the acute and parasitemic recurrence stages. Compared to CMs, RPMs and NCMs expanded rapidly during the recurrence stage. Reduction of NCMs with a low-dose of CLs during the recurrence stages significantly elevated the \u003cem\u003eP. chabaudi\u003c/em\u003e parasitemia. Moreover, during both the acute and recurrent phases of \u003cem\u003eP. chabaudi\u003c/em\u003e infection, CCR2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e infected mice exhibited no significant changes in the percentages of splenic NCMs (Live/Dead\u003csup\u003e\u0026minus;\u003c/sup\u003eLy6G\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003eint\u003c/sup\u003eLy6C\u003csup\u003e\u0026minus;\u003c/sup\u003e) and CMs (Live/Dead\u003csup\u003e\u0026minus;\u003c/sup\u003eLy6G\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003eint\u003c/sup\u003eLy6C\u003csup\u003e+\u003c/sup\u003e) within the monocyte population, when compared to their littermate controls. Thus, these findings suggest that the expansion of NCMs in spleen may not be directly attributed to the conversion of CMs during \u003cem\u003eP. chabaudi\u003c/em\u003e infection. Taken together, our findings indicate that NCMs play a potential role in controlling parasitemic recurrence, which deepens our understanding of the determinants of immunity against \u003cem\u003ePlasmodium\u003c/em\u003e infections.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003eC57BL/6 mice were purchased from GemPharmatech (Nanjing, Jiangsu, China). CCR2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (B6.129S4-\u003cem\u003eCcr2\u003c/em\u003e\u003csup\u003e\u003cem\u003etm1Ifc\u003c/em\u003e\u003c/sup\u003e/J) were obtained from professor Li Tang (Laboratory of Hepatology and Immunology, State Key Laboratory of Proteomics, Beijing, China). All mice were bred and maintained in our pathogen-free animal facility. Only female mice, aged 6\u0026ndash;8 weeks, were used in our study. All mouse procedures were approved by the Animal Ethics Committee of the Army Medical University (Third Military Medical University) Institute of Medical Research, under the approval number AMUWEC20230344.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eParasite and infection\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003ePlasmodium chabaudi chabaudi AS\u003c/em\u003e (\u003cem\u003eP. chabaudi\u003c/em\u003e) was maintained in our laboratory. For infection studies, female mice were challenged intraperitoneally (i.p.) with 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e \u003cem\u003eP. chabaudi\u003c/em\u003e-parasitized red blood cells (pRBCs). Parasitemia was assessed by examining Giemsa-stained thin blood smears every other day following infection.\u003c/p\u003e\n\u003ch3\u003escRNA-seq analysis\u003c/h3\u003e\n\u003cp\u003eThe scRNA-seq data was sourced from our previously published research [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and the raw data has been deposited in the Gene Expression Omnibus (GEO) database under the accession number GSE192930. To analyze these scRNA-seq data, we employed the Seurat package (version 4.4.0) within the R programming environment (version 4.3.2) to perform unsupervised clustering of individual cells. Specifically, the Uniform Manifold Approximation and Projection (UMAP) method was employed for dimensionality reduction. In our previously published article [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], we classified spleen cells from \u003cem\u003eP. chabaudi\u003c/em\u003e-infected mice into eight distinct populations: B cells, dendritic cells (DCs), RPMs, monocytes, neutrophils, NK cells, platelets, and T cells. In the current study, we selected RPMs and monocytes for further re-clustering analysis. General cell types were annotated by examining the expression of cell type-specific marker genes, along with genes that contribute to the heterogeneity observed within clusters. The genes of \u003cem\u003eAdgre1\u003c/em\u003e, \u003cem\u003eSpic\u003c/em\u003e, and \u003cem\u003eVcam1\u003c/em\u003e were used to delineate RPMs; \u003cem\u003eItgam\u003c/em\u003e, \u003cem\u003eCsf1r\u003c/em\u003e, \u003cem\u003eLy6c2\u003c/em\u003e, and \u003cem\u003eCcr2\u003c/em\u003e for CMs; and \u003cem\u003eItgam\u003c/em\u003e, \u003cem\u003eCsf1r\u003c/em\u003e, and \u003cem\u003eCx3cr1\u003c/em\u003e for NCMs. Additionally, utilizing the Seurat package in R (version 4.4.0), we conducted differential gene expression analysis to compare the expression profiles between the two groups.\u003c/p\u003e\n\u003ch3\u003eFlow cytometry analysis\u003c/h3\u003e\n\u003cp\u003eThe following antibodies were used in our experiments for flow cytometry analysis: anti-mouse/human CD11b (PerCP/Cyanine5.5, clone M1/70, BioLegend), anti-mouse Ly6G (APC, clone 1A8, BioLegend), anti-mouse Ly6C (PE, clone HK1.4, BioLegend), anti-mouse F4/80 (FITC, clone BM8, BioLegend), anti-mouse CD11c (APC, 117310, clone N418, BioLegend). All above antibodies were used at a 1:100 dilution. LIVE/DEAD\u0026trade; Fixable Violet Staining kit (Invitrogen) was used at a 1:250 dilution to exclude the dead cells. For flow cytometry staining, spleens were collected at the indicated days, and single-cell suspensions of splenocytes were obtained. For surface staining, 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e splenocytes were firstly incubated with anti-CD16/32 antibodies (clone 93, BioLegend) to block Fc receptors. Then the splenocytes were stained with the antibodies of interest for 45 min. The cells were acquired on a FACSCanto II instrument (BD Bioscience, SanJose, CA, United States) and FlowJo v10 software (Tree Star Inc.) was used to analyzed the data. CD11b, Ly6G, F4/80, Ly6C and CD11c were used to define the different cell populations, including the neutrophils, RPMs, CMs, NCMs, and DCs.\u003c/p\u003e\n\u003ch3\u003eLow-dose CLs-based treatment\u003c/h3\u003e\n\u003cp\u003eC57BL/6 mice were injected intravenous (i.v.) with a single dose of 6.5 mg/kg body weight of clodronate liposomes (CLs) or PBS-loaded control liposomes (PBSL) (Liposoma BV) in a final volume of 200\u0026micro;l when the parasitemia in acute stage declined almost to zero. The impact of liposomes was evaluated 24 hours post-injection through flow cytometry analysis of splenic phagocytic cells, including NCMs (Live/Dead\u003csup\u003e\u0026minus;\u003c/sup\u003eLy6G\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003eint\u003c/sup\u003eLy6C\u003csup\u003e\u0026minus;\u003c/sup\u003e), CMs (Live/Dead\u003csup\u003e\u0026minus;\u003c/sup\u003eLy6G\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003eint\u003c/sup\u003e Ly6C\u003csup\u003e+\u003c/sup\u003e), RPMs (Live/Dead\u003csup\u003e\u0026minus;\u003c/sup\u003eLy6G\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11b\u003csup\u003e\u0026minus;\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e), DCs (Live/Dead\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11c\u003csup\u003ehi\u003c/sup\u003e), and neutrophils (Live/Dead\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eThe spleens were surgically removed from C57BL/6 mice on day 0, 16 post-infection, with low-dose CLs or control PBSL treatment. Spleens were promptly snap-frozen and cryosectioned into 8-micron thick slices, which were then mounted onto glass slides. The sections were fixed with 4% paraformaldehyde in PBS for 30 minutes at room temperature (RT). Subsequently, they were washed three times with PBS and permeabilized with 0.1% saponin in PBS for 10 minutes at RT. The fixed sections were incubated overnight at 4 ℃ with primary antibodies diluted in a blocking solution, including anti-CD45R (B220) monoclonal antibody labeled with Alexa Fluor 488 (53-0452-82, Invitrogen, 1:25), and either anti-SIGN-R1 (05PB1435, BMA BIOMEDICALS, 1:400) or anti-MoMA-1 (11PB1616, BMA BIOMEDICALS, 1:400). After washing with PBS, the sections were incubated for 1 hour at RT with Streptavidin conjugated to Alexa Fluor\u0026trade; 647 (S21374, Invitrogen, 1:100). Images were captured using the LSM 800 microscope system and analyzed with ZEN Imaging Software 3.4 (Zeiss). The Integrated Density (IntDen) of SIGN-R1 and MoMA-1 was quantified using ImageJ v2.1. Cells were stained with anti-CD45R (B220) for B cells, anti-SIGN-R1 for marginal zone macrophages (MZM) and anti-MoMA-1 for marginal metallophilic macrophages (MMM).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIdentification of CCR2 mice\u003c/h3\u003e\n\u003cp\u003eThe offspring of the CCR2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were identified by Polymerase Chain Reaction (PCR) and flow cytometry. For PCR analysis, the absence of CCR2 sequence were identified by using genomic DNA isolated from tail tissue. The primers set were FW1 (5\u0026rsquo;-CCACAGAATCAAAGGAAATGG-3\u0026rsquo;), RV1(5\u0026rsquo;-CACAGCATGAACAATAGCCAA G-3\u0026rsquo;), RV2 (5\u0026rsquo;-CCTTCTATCGCCTTCTTGAC G). For flow cytometry analysis, inflammatory monocytes (Ly6C\u003csup\u003ehi\u003c/sup\u003e monocytes, Ly6G\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehi\u003c/sup\u003e) were identified to differentiate the CCR2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice from their wild-type littermates, as previously reported [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are given as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SD) unless otherwise stated. Statistical analysis was performed using GraphPad Prism version 8.0 (GraphPad Software, La Jolla, CA, United States). Statistically significant differences between the two groups were determined using the unpaired Student\u0026rsquo;s t-test, one-way ANOVA or two-way ANOVA. Statistical significance is indicated as follows: *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and 'ns' indicates 'not significant'.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eSingle cell analysis reveals the dynamic change of monocytes and macrophages between acute and recurrence stages\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur previous findings indicated that phagocytic cells participate in controlling parasitemic recurrence. To further investigate which type of phagocytic cells prevent this recurrence, scRNA-seq data from our previous study was further analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. We extracted RPMs and monocytes and conducted re-clustering analysis. With a resolution of 0.8, the cells were divided into 13 clusters (Additional file 1: Fig. S1a). We used the genes \u003cem\u003eAdgre1\u003c/em\u003e (encoding F4/80), \u003cem\u003eSpic\u003c/em\u003e (encoding Spi-C), \u003cem\u003eVcam1\u003c/em\u003e (encoding VCAM1), \u003cem\u003eItgam\u003c/em\u003e (encoding CD11b), \u003cem\u003eCsf1r\u003c/em\u003e (encoding CD115), \u003cem\u003eLy6c2\u003c/em\u003e (encoding Ly6C), \u003cem\u003eCcr2\u003c/em\u003e (encoding CCR2), and \u003cem\u003eCx3cr1\u003c/em\u003e (encoding CX3CR1) to define the cell subpopulations. Spi-C is a characteristic transcription factor for RPMs, with F4/80 and VCAM1 typically exhibiting elevated expression levels on their surface [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Previous study has established that murine CMs are defined as CD11b\u003csup\u003e+\u003c/sup\u003eCD115\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehi\u003c/sup\u003eCCR2\u003csup\u003ehi\u003c/sup\u003eCX3CR1\u003csup\u003elow\u003c/sup\u003e, whereas NCMs are defined as CD11b\u003csup\u003e+\u003c/sup\u003eCD115\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003elow\u003c/sup\u003eCX3CR1\u003csup\u003ehi\u003c/sup\u003eCCR2\u003csup\u003elow\u003c/sup\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, we utilized the genes \u003cem\u003eItgam\u003c/em\u003e, \u003cem\u003eCsf1r\u003c/em\u003e, \u003cem\u003eLy6c2\u003c/em\u003e, \u003cem\u003eCcr2\u003c/em\u003e, and \u003cem\u003eCx3cr1\u003c/em\u003e to differentiate between CMs and NCMs. After conducting the clustering analysis, monocytes and macrophages were found to consist of three distinct subsets: CMs (\u003cem\u003eItgam\u003c/em\u003e, \u003cem\u003eCsf1r\u003c/em\u003e, \u003cem\u003eLy6c2\u003c/em\u003e, \u003cem\u003eCcr2\u003c/em\u003e), NCMs (\u003cem\u003eItgam\u003c/em\u003e, \u003cem\u003eCsf1r\u003c/em\u003e, \u003cem\u003eCx3cr1\u003c/em\u003e), and RPMs (\u003cem\u003eAdgre1\u003c/em\u003e, \u003cem\u003eSpic, Vcam1\u003c/em\u003e). As shown in Fig. S1b, populations 3 and 8 correspond to CMs, population 6 corresponds to NCMs, and the populations 2, 4, 9, 10, 11 are classified as RPMs (Additional file 1: Fig. S1b; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-d). The percentage of CMs substantially increased during the acute stage and subsequently decreased during the recurrent phase. However, a contrasting trend was observed in the percentages of NCMs and RPMs, with a decline during the acute stage and subsequent increase during the recurrent phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Since splenic RPMs are not crucial for controlling \u003cem\u003eP. chabaudi\u003c/em\u003e infection [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], we postulated that NCMs, which markedly increased during the recurrence stage, potentially play a pivotal role in suppressing parasite recurrence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePlasmodium\u003c/b\u003e \u003cb\u003einfection induces NCMs expansion in the transition of acute to recurrence stages\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further explore the potential role of NCMs in controlling parasitemic recurrence, the changing trend in NCMs frequency and number was analyzed using flow cytometry. Specifically, splenocytes were collected from naive mice as well as mice infected with \u003cem\u003eP. chabaudi\u003c/em\u003e at days 8, 12, and 16 post-infection for this analysis. Consistent with the scRNA-seq data, the proportion of NCMs (Live/Dead\u003csup\u003e\u0026minus;\u003c/sup\u003eLy6G\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003eint\u003c/sup\u003eLy6C\u003csup\u003e\u0026minus;\u003c/sup\u003e) in either the splenocytes or monocytes continued to decline during the acute stage (day 8 post-infection), but increased rapidly during the recurrent phase (day 16 post-infection) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c). Although the number of splenic NCMs on day 8 post-infection is higher than that of na\u0026iuml;ve mice, which contrasts with the trend observed in NCMs frequency, the number of NCMs remains significantly elevated during the recurrence stage (days 12 and 16 post-infection) compared to days 0 and 8 post-infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). These results further suggest that NCMs may play a potential role in controlling parasitemic recurrence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eNCMs, but not other phagocytes, were selectively reduced after low-dose CLs treatment in\u003c/b\u003e \u003cb\u003eP. chabaudi\u003c/b\u003e \u003cb\u003erecurrence\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the role of NCMs in controlling recurrent infections of \u003cem\u003eP. chabaudi\u003c/em\u003e, it would be ideal to use conditional knockout mice that allow for inducible depletion of NCMs. However, such mice are not commercially available. Alternatively, the small-molecule inhibitor AZD8797 has been used to selectively reduce the population of Ly6C\u003csup\u003elow\u003c/sup\u003e NCMs [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, we found that the administration of AZD8797 did not significantly deplete NCMs during the recurrent phase of \u003cem\u003eP. chabaudi\u003c/em\u003e infection (data not shown). Therefore, alternative approaches are needed to specifically target and deplete NCMs. CLs are commonly used to specifically deplete phagocytes, including macrophages, monocytes, and DCs, in various organs and tissues [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, the effectiveness of this depletion varies with the dose of CLs, with low doses demonstrating a more selective impact on specific phagocyte subsets. Therefore, we investigated whether the administration of low-dose CLs could reduce NCMs during the recurrent phase of \u003cem\u003eP. chabaudi\u003c/em\u003e infection.\u003c/p\u003e \u003cp\u003eSubsequently, we examined the frequencies and numbers of major phagocytic cell populations in spleen 24 hours after administering the low-dose CLs. These populations included RPMs (Live/Dead\u003csup\u003e\u0026minus;\u003c/sup\u003eLy6G\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11b\u003csup\u003e\u0026minus;\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e), CMs (Live/Dead\u003csup\u003e\u0026minus;\u003c/sup\u003eLy6G\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003eint\u003c/sup\u003eLy6C\u003csup\u003e+\u003c/sup\u003e), NCMs (Live/Dead\u003csup\u003e\u0026minus;\u003c/sup\u003eLy6G\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003eint\u003c/sup\u003eLy6C\u003csup\u003e\u0026minus;\u003c/sup\u003e), DCs (Live/Dead\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11c\u003csup\u003ehi\u003c/sup\u003e) and neutrophils (Live/Dead\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e). As anticipated, intravenous administration of low-dose CLs on day 14 post-infection did not significantly affect the frequency and number of RPMs, CMs, DCs or neutrophils, but significantly reduced the frequency and number of splenic NCMs in infected mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c). Given that previous studies indicated low-dose CLs specifically target the depletion of MZM and MMM [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], we assessed both splenic MZM and MMM in parasite-infected mice treated with or without low-dose CLs using immunofluorescence assays. Remarkably, both MZM and MMM were absent in the infected mice, regardless of whether they were treated with low-dose CLs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, e). The depletion of MZM and MMM in the spleens of \u003cem\u003eP. chabaudi\u003c/em\u003e-infected mice aligns with previous findings [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Thus, our data suggest that low-dose CLs can effectively reduce the total number of NCMs during the recurrent phase of \u003cem\u003eP. chabaudi\u003c/em\u003e infection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLow-dose CLs treatment after acute parasitemia results in\u003c/b\u003e \u003cb\u003eP. chabaudi\u003c/b\u003e \u003cb\u003erecurrence\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBased on the aforementioned findings, we employed a dosage of 6.5 mg/kg of CLs to investigate the effect of NCMs reduction on parasitemic recurrence of \u003cem\u003eP. chabaudi\u003c/em\u003e infection. Remarkably, intravenous administration of a low-dose of CLs in the recurrent phase (day 14 post-infection) significantly elevated the parasitemia levels, which were much higher than the peak in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). However, no significant effect on the survival rate was observed after the administration of low-dose CLs in the recurrence stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Thus, our data strongly suggests a potential role for NCMs in preventing parasitemic recurrence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe transition of CMs was not essential for the development of NCMs during the\u003c/b\u003e \u003cb\u003eP. chabaudi\u003c/b\u003e \u003cb\u003erecurrence\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIt has been observed that CMs from the blood continuously replenish the monocyte pool in the spleen and also differentiate into NCMs [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Given this, we wondered whether the fluctuation in the frequency of spleen NCMs following \u003cem\u003eP. chabaudi\u003c/em\u003e infection is associated with the conversion of CMs. Since CCR2 is pivotal in facilitating the egression of CMs from the bone marrow into the bloodstream, mice deficient in CCR2 exhibit a deficiency in the majority of CMs [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Therefore, we used CCR2-deficient mice to test our hypothesis. Firstly, we referred to previous study and utilized inflammatory monocyte (Live/Dead\u003csup\u003e\u0026minus;\u003c/sup\u003eLy6G\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehi\u003c/sup\u003e) detection to validate the CCR2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Consistent with previous finding, the percentage of splenic inflammatory monocytes in uninfected CCR2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice was significantly lower than that in uninfected littermate controls (Additional file 2: Fig. S2b, c). Notably, during \u003cem\u003eP. chabaudi\u003c/em\u003e infection, there were no significant differences in the percentages of CMs and NCMs in the splenic monocytes between CCR2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice and their littermate controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Moreover, on days 0 and 7 post-infection, CCR2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice exhibited a significantly reduced number of splenic CMs compared to littermate controls. While the number of NCMs in uninfected CCR2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice was significantly higher than in their littermate counterparts, it was significantly lower than that of littermate controls on day 7 post-infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). However, during the recurrent phase of \u003cem\u003eP. chabaudi\u003c/em\u003e infection, no significant differences were observed between CCR2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice and littermate controls in terms of either the frequency or the number of splenic NCMs and CMs. Thus, these findings suggest that the increasing frequency and number of NCMs during the recurrent phase of \u003cem\u003eP. chabaudi\u003c/em\u003e infection may not be attributable to the transition of CMs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePreventing the parasitemic recurrence is crucial in blocking malaria transmission [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, our understanding of how immune cells control parasitic recurrence remains limited. Recent research has found that NCMs significantly expand in chronic rodent and human \u003cem\u003eplasmodium\u003c/em\u003e infections [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]; however, the underlying biological significance of this phenomenon remains elusive. In the current study, our findings revealed that the frequency of NCMs significantly decreased in the acute stage but underwent a rapid increase during the recurrent phase of \u003cem\u003eP. chabaudi\u003c/em\u003e infection. Notably, the reduction of NCMs during the recurrent phase led to a rapid increase in parasitemia, indicating their potential role in controlling parasitemic recurrence. Furthermore, using CCR2-deficient mice, we found that the increased NCMs frequency and number during \u003cem\u003eP. chabaudi\u003c/em\u003e recurrence is not due to the conversion of CMs. These findings may highlight the specialized protective role of NCMs in preventing post-acute parasitemic recurrence, enhance our understanding of the host's ability to control chronic malaria infection, and provide theoretical support for blocking malaria transmission.\u003c/p\u003e \u003cp\u003eOur study still has several limitations. Despite the proven efficacy of low-dose CLs in reducing NCMs, this approach remains inconclusive in establishing the definitive protective role of NCMs in preventing malaria recurrence. Previous studies have shown that other phagocytes, particularly neutrophils, contribute to protection against \u003cem\u003ePlasmodium\u003c/em\u003e infection via antibody-dependent respiratory bursts [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, our findings indicate that intravenous administration of low-dose CLs has no impact on the splenic neutrophil frequency and number. Notably, ablation of the Nr4a1 super-enhancer subdomain, referred to as E2, can effectively deplete NCMs [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, we are still devoid of strategies to generate conditional knockout mice that selectively deplete NCMs during the recurrent phase of \u003cem\u003eP. chabaudi\u003c/em\u003e infection. Recent studies have highlighted the high-affinity Cx3cr1 small-molecule inhibitor, AZD8797, in selectively diminishing the population of Ly6C\u003csup\u003elow\u003c/sup\u003e NCMs [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Consequently, we have adopted the method described in this literature, utilizing the Cx3cr1 inhibitor, AZD8797, to specifically deplete NCMs, aiming to clearly elucidate the pivotal role of NCMs in regulating the recurrent phase of \u003cem\u003eP. chabaudi\u003c/em\u003e infection. Nevertheless, our preliminary experiments revealed that the frequency and number of NCMs remained unaffected following the administration of AZD8797 during the recurrent phase of \u003cem\u003eP. chabaudi\u003c/em\u003e infection (data not shown). Previous study has indicated that intermediate monocytes may play a crucial role in controlling \u003cem\u003ePlasmodium\u003c/em\u003e infections by phagocytosing and clearing infected erythrocytes [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Therefore, further research is essential to conclusively clarify the protective roles of NCMs and intermediate monocytes in preventing malaria recurrence.\u003c/p\u003e \u003cp\u003eMeanwhile, the effective mechanism underlying the suppression of parasitemic recurrence by NCMs remain unclear. Previous research has predominantly centered on the function of NCMs in clearing debris through Fcγ-mediated phagocytosis in vessels and monitoring endothelial cell integrity. Furthermore, neutrophils in peripheral blood, rather than monocytes, can mediate phagocytosis of sporozoites through Fcγ-receptor (FcγR). However, it remains unclear whether different subsets of monocytes in the spleen can exert immune-protective effects through FcγR [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Therefore, examining the expression of FcγR on various subsets of splenic monocytes will aid in understanding their roles in controlling \u003cem\u003ePlasmodium\u003c/em\u003e infection. Additionally, a recent study revealed that NCMs robustly express the immune checkpoint molecule PD-L1 (CD274) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Notably, under inflammatory conditions, PD-L1\u003csup\u003e+\u003c/sup\u003e NCMs exhibit immunomodulatory functions, promoting T cell apoptosis in tertiary lymphoid organs (TLOs) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Moreover, NCMs exhibit heightened production of inflammatory cytokines in response to viral and nucleic acid stimuli, mediated by TLR7 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. They also trigger the recruitment and activation of innate immune cells, including NK cells and neutrophils, through TNF-α-mediated upregulation of E-selectin on endothelial cells [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Considering the intricate role of NCMs in the immune response, we still lack clues regarding their protective effects against malaria recurrence. Previous studies have demonstrated that monocytes can exert immune protection against blood-stage malaria infection by phagocytosing infected erythrocytes. Additionally, monocytes may also contribute to immune defense through antibody-dependent cellular inhibition [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, this study has not yet definitively established whether NCMs also possess such protective effects. Therefore, further research is necessary to clarify the protective mechanisms of NCMs during the recurrent phase of \u003cem\u003eP. chabaudi\u003c/em\u003e infection.\u003c/p\u003e \u003cp\u003eCMs derive from myeloid-committed progenitors residing in the bone marrow (BM) and subsequently traverse into the circulation via a CCR2-dependent mechanism [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Conversely, the precise origin of NCMs continues to be enigmatic. Recent studies have revealed that NCMs can be derived from CM transitions [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. CMs and NCMs have biological interconnections and show minimal differences in chromatin organization [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. However, our findings demonstrate that the percentage of splenic NCMs and CMs within the monocyte population exhibited no significant differences between CCR2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice and their control littermates during the \u003cem\u003eP. chabaudi\u003c/em\u003e infection, partially aligning with prior research findings [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. These findings suggest that the transition to NCMs occurs independently of CCR2, a molecule exclusively expressed by CMs, in the context of \u003cem\u003eP. chabaudi\u003c/em\u003e infection. The underlying mechanism may be attributed to trapped CMs in the BM of CCR2-deficient mice, resulting in a marked depletion of CMs [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Additionally, the transformation of CMs into NCMs appears to be less efficient in aged mice [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. These observations suggest that the origin of NCMs is complex. Consistent with prior report, our findings also reveal a significant increase in the number of CMs in the spleen of CCR2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice during the later stages of \u003cem\u003eP. chabaudi\u003c/em\u003e infection [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These observations imply that, apart from CMs, other immune cells may also contribute to replenishing splenic CMs reservoir during chronic \u003cem\u003eP. chabaudi\u003c/em\u003e infection. Furthermore, our research revealed that the number of NCMs in the spleens of CCR2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e uninfected mice was significantly higher than in their control littermates. However, the underlying molecular mechanisms driving this phenomenon remain elusive.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe significance of NCMs in both health and disease contexts is increasingly recognized; nonetheless, their specific contribution to malarial infection remains undefined. In the present study, we demonstrated that NCMs, which expanded during the recurrent phase of \u003cem\u003eP. chabaudi\u003c/em\u003e infection, have the potential role in controlling chronic malaria infection. Notably, this expansion may not originate from the transition of CMs. Therefore, our findings not only contribute to understanding how innate immune cells control the recrudescence of \u003cem\u003eplasmodium\u003c/em\u003e infection, but also lay a theoretical foundation for the prevention of malaria transmission.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003escRNA-seq\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esingle-cell RNA sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRPMs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ered pulp macrophages\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCMs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eclassical monocytes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNCMs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enon-classical monocytes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCLs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eclodronate liposomes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBSL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePBS-loaded control liposomes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMZM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emarginal zone macrophages\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMMM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emarginal metallophilic macrophages\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eP. chabaudi\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003ePlasmodium chabaudi chabaudi AS\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ei.p.\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eintraperitoneally\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003epRBCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eparasitized red blood cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUMAP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUniform Manifold Approximation and Projection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edendritic cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTh1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eT helper1 cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTfh\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eT follicular helper cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIntDen\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIntegrated Density\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTLOs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etertiary lymphoid organs\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebone marrow.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Jiayin Biotechnology Ltd. (Shanghai, China), for the assistance with scRNA assay. We would like to thank Editage (www.editage.cn) for English language editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 82172296), the Natural Science Foundation of Chongqing (No. CSTB2023NSCQ-MSX0788).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the conclusions of this article are included within the article. Data of scRNA used in this study are publicly available from the GEO database at www.ncbi.nlm.nih.gov/geo/ under accession number GSE192930.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTL and WX conceived the experiment and wrote the paper. JF, SC, YG, YF, SG, XL, HL and JZ performed the research and analyzed the data. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal studies were reviewed and approved by the Animal Ethics Committee of the Third Military Medical University Institute of Medical Research, Chongqing, China (ethical approval number: AMUWEC20230344).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of Pathogenic Biology, Army Medical University (Third Military Medical University), Chongqing, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eClinical Laboratory Diagnostic Center, General Hospital of Xinjiang Military Region, Urumqi, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003eInstitute of Immunology, Army Medical University (Third Military Medical University), Chongqing, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e4\u003c/sup\u003eKey Laboratory of Extreme Environmental Medicine, Ministry of Education of China, Chongqing, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWorld Health Organization. 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Immunity. 2017;46:849\u0026ndash;62. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.immuni.2017.04.018\u003c/span\u003e\u003cspan address=\"10.1016/j.immuni.2017.04.018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Malaria parasite, NCMs, recurrence, CCR2","lastPublishedDoi":"10.21203/rs.3.rs-5928249/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5928249/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eFrequent recurrence is responsible for persistent \u003cem\u003ePlasmodium\u003c/em\u003e infection after the acute stage. Our previous study demonstrated that phagocytic cells are essential for controlling \u003cem\u003ePlasmodium chabaudi chabaudi AS\u003c/em\u003e (\u003cem\u003eP. chabaudi\u003c/em\u003e) recurrence. Nevertheless, the specific type of phagocytic cells involved in controlling \u003cem\u003eP. chabaudi\u003c/em\u003e recurrence, as well as their underlying molecular mechanisms of action, remain elusive.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eSingle-cell RNA sequencing (scRNA-seq) was employed to analyze splenic phagocytic cells during both the acute and recurrent phases of \u003cem\u003eP. chabaudi\u003c/em\u003e infection. The frequencies of red pulp macrophages (RPMs), classical monocytes (CMs), and non-classical monocytes (NCMs) were detected by flow cytometry. Low-dose clodronate liposomes (CLs) and CCR2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were used to investigate the protective role and origin of NCMs.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eUsing scRNA-seq, we found that NCMs declined during the acute stage of \u003cem\u003eP. chabaudi\u003c/em\u003e blood-stage infection, and then expanded rapidly in the recurrence stage. The changing trend of NCMs was confirmed by flow cytometry. To explore the potential role of NCMs in controlling parasitemic recurrence, NCMs were reduced by a low-dose of CLs during the recurrence stage, which significantly elevated the \u003cem\u003eP. chabaudi\u003c/em\u003e parasitemia. Additionally, no significant difference in the proportion of splenic NCMs or CMs within the monocyte population was observed between the infected CCR2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice and their control littermates, suggesting that the transition from CMs to NCMs may not occur in this model.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIn summary, our results indicate that NCMs potentially play a protective role in preventing malarial parasitemic recurrence, offering valuable insights into immune-based interventions against \u003cem\u003ePlasmodium\u003c/em\u003e infection and potentially contributing to the prevention of malaria transmission.\u003c/p\u003e","manuscriptTitle":"The potential role of non-classical monocytes in preventing malarial parasitemic recurrence in a mouse model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-03 17:00:04","doi":"10.21203/rs.3.rs-5928249/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"01b70484-a625-4852-a3b9-ab59571a43ba","owner":[],"postedDate":"February 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-13T08:54:44+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-03 17:00:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5928249","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5928249","identity":"rs-5928249","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}