Introduction

Lower respiratory tract infections (LRTIs), with outbreaks mostly occurring in the fall and winter, are the major cause of morbidity and mortality in children. Among LRTIs, community-acquired pneumonia (CAP) is the most common cause of hospitalization/death for children in developed/developing countries, respectively1,2. In children over the age of five, MPP accounts for over 40% of cases of CAP3. After being inhaled, MPP easily and closely attaches to ciliated cells of the respiratory epithelium via special attaching organelles due to the absence of a cellular wall structure, and thereby exchange chemicals/proteins with host cells efficiently4. The most common clinical presentations of MPP are fever, dyspnea, and cough, sometimes accompanied by runny nose, nasal congestion, chest pain, wheezing, and pharyngitis5,6, in addition to a variety of extrapulmonary manifestations like gastrointestinal and skin disorders7. Massive pleural effusions may also be linked to severe instances of MPP. Despite that tetracyclines and fluoroquinolones are effective in reducing the duration of fever and respiratory symptoms of MPP, many questions regarding their safety and effectiveness remain undetermined8,9.

It has been demonstrated that both the innate and adaptive immune systems participate in MPP infections, and thus the magnitude of immune responses highly correlate to the disease severity. Upon MPP infection, alveolar macrophages ingest the pathogen and present it to cognate T cells, eventually triggering the production of cytokines and chemokines, which will recruit more immune cells into the infection sites to amplify the inflammatory responses. The M1 and M2 are two major types of macrophages. LPS and IFN-γ induce the differentiation of M1 macrophages, which produce chemokines like CXCL1, CXCL5, CXCL9, CXCL10, CCL3, and CCL19, as well as cytokines including IL-6, IL-8, IL-10, IL-1β, and TNF-α10,11. By contrast, M2 macrophages, normally differentiated in the presence of IL-4, produce a distinct set of chemokines, such as CCL1, CCL14, CCL17, and CCL24. M1-related chemokines predominate during the acute phase of Mycoplasma pneumoniae infection in children. However, the role of M1-related chemokines during this phase of infection remains unclear11. Amongst these soluble factors, CXCL10, a member of the CXC chemokine family produced by M1 macrophages, plays a critical role in the recruitment and activation of neutrophils, NK cells and lymphocytes. As such, serum levels of CXCL10 in children have been shown to correlate with MPP infection and could serve as a useful biomarker for monitoring MPP progression in clinical settings, offering higher specificity and sensitivity compared to other biomarkers such as CRP and LDH12,13. Nevertheless, how the production of CXCL10 is modulated in children with MPP remains elusive and thus warrants additional investigations.

It has been reported that Dickkopf-1 (DKK1) may be effective in reducing the MPP-induced massive secretion of mucin 5AC (MUC5AC) by inhibiting the JAK/STAT signaling pathway in a mouse model of MPP14,15. Moreover, IFN-γ induces CXCL10 through activation of the JAK/STAT1 signaling in many immune disorders16,17. STAT1 and CXCL10 promote the polarization of M1-type macrophages18. Therefore, we speculated that IFN-γ may promote CXCL10 expression by M1-type macrophages through the JAK/STAT1 pathway, and thereby exacerbates inflammatory responses in the lungs of patients with MPP. In this study, we collected peripheral blood and BALF in children with MPP, and analyzed the correlation between CXCL10 levels and other immunological/clinical parameters. In addition, we also deciphered pathways underlying the IFN-γ-induced CXCL10 productions in macrophages in vitro. Our findings may be helpful for the diagnosis and tailored treatment/management of children with MPP.

Methods

Patients

The study was a case-control study with α = 0.05, β = 0.1, and the sample sizes for the case and experimental groups were determined through calculations using PASS software. A total of 122 hospitalized children with a diagnosis of MPP in the Department of Respiratory Medicine at Children’s Hospital of Soochow University during March to November 2023, among whom 63 (51.64%) were male and 59 (48.36%) were female. 48 cases were categorized as group A to determine the relationship between serum CXCL10 levels and pathogen loads in nasopharyngeal aspirates as well as other clinical parameters within 24 h of hospitalization; 74 patients with IFN-γ and CXCL10 levels in BALF were selected as group B to investigate the correlation of IFN-γ/CXCL10 and immunoinflammatory injury in the lungs. The ages of the children in groups A and B ranged from 1 to 15 years. Peripheral blood from 40 children who had physical examinations at the Department of Child Health outpatient clinic and BALF from 14 children who underwent emergency surgery in the Ear Nose and Throat (ENT) Department of our hospital within the same period as mentioned above served as control samples. Children in the control group were in good health over the course of last four weeks before samples were taken, and had no history of drug use, respiratory conditions such as airway deformities, and cardiac, hepatic, renal, hematologic, or metabolic illnesses.

Diagnostic criteria

(1) Meets the diagnosis of CAP19,20. The diagnostic criteria for CAP are as follows: (i) The presence of clinical symptoms such as cough, sputum production, fever, and shortness of breath; (ii) The presence of fixed pulmonary wet rales and/or signs of pulmonary consolidation; (iii)Chest X-ray showing patchy infiltrative shadows or interstitial changes, with or without pleural effusion. (2) Meets the diagnostic criteria for Mycoplasma pneumonia infection: positive MPP-specific immunoglobulin (IgM) in peripheral blood and MPP DNA in nasopharyngeal aspirate or BALF ≥ 1.0 × 104 copies/ml as determined by qPCR.

Exclusion criteria

(1) Recurrent respiratory infections, defined as eight or more documented respiratory tract infections per year in preschool children (up to three years old) and six or more in children older than three years, in the absence of any pathological condition underlying recurrent infections21; (2) Clinical signs and supporting data of typically bacterial pneumonia; (3) High-risk factors or clinical and imaging features of fungal pneumonia; (4) Positive antigen or nucleic acid test for NPA or BALF virus; (5)Presence of congenital disorders or birth defects; (6) Respiratory diseases, including metabolic, cardiac, hepatic, renal, and other systemic abnormalities; (7) Being involved in an experimental study involving a drug or device treatment.

Collection of clinical data

The electronic medical record system was used to gather the children’s clinical data, including the age, sex, cough, fever duration/peaks, number of hospital days, pulmonary symptoms, and imaging, etc. White blood cells (WBC), neutrophil percentages (N%), platelets (PLT), lactate dehydrogenase (LDH), C-reactive protein (CRP), immunoglobulins (IgA/IgG/IgM), lymphocyte subpopulations, and so on were measured in the laboratory.

Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis

After the extraction of total cellular RNA from cultured cells using Trizol reagent (Invitrogen), 1 µg of RNA was reverse transcribed to cDNA with the M-MLV Reverse Transcriptase (ELK Biotechnology), and then qPCR was conducted by using the QuFast SYBR Green PCR Master Mix (ELK Biotechnology).

The primer sequences were as follows: CXCL10, Antisense 5′-GTAAATTCTTGATGGGCCTTCG-3′, and Sense5′-CCATTCTGATTTGCTGCCTTAT-3′; β-Actin, Antisense5′-CTGGAAGGTGGACAGCGAGG-3′, and Sense 5′- TGACGTGGACATCCGCAAAG-3′; siSTAT1, Antisense 5′-TCTACAGAGCCCACTATCCGAG-3′, and Sense 5′- ACTTTCCCTGACATCATTCGC-3′.

Protein chip

Peripheral blood samples from the refractory M. pneumoniae pneumonia (RMPP), non-refractory M. pneumoniae pneumonia (NRMPP), and healthy patients were collected. Peripheral blood protein microarray high-throughput protein screening technology and results provided by Ray Biotech Co., Ltd. (Guangzhou, China).

Cell culture

THP-1 cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% sodium pyruvate, 1% HEPES, and 50 mM β-mercaptoethanol at 37 °C in a humidified atmosphere of 5% CO2. Macrophages were obtained by culturing THP-1 cells with phorbol ester (PMA, 50 ng/ml) for 24 h. Subsequently, these THP-1 derived M1-type macrophages were stimulated with different concentrations of IFN-γ, and the expression levels of CXCL10 were detected at different time points (3, 6, and 9 h). CD4+ T cells were isolated from healthy human peripheral blood by magnetic beads (the Direct Human CD4+ T Cell Isolation Kit, STEMCELL Technologies, Vancouver, Canada), and were stimulated in vitro with anti-CD3/28 in the presence of IL-2 & IL-12 for 4 days to induce the differentiation toward Th1 cells.

ELISA

Levels of CXCL10 and IFN-γ in the peripheral blood and/or BALF of MPP and control groups were measured by precise ELISA kits in accordance with the manufacturer’s instructions (Asahi Kok Hing Biotech Co., Ltd., Suzhou, China).

Flow cytometry

Percentages of macrophages, lymphocytes, and neutrophils in the BALF of children were determined by flow cytometer (Beckman Coulter, USA). The following antibodies were used: anti-CD16 (clone GRM1), anti-CD64 (clone 10.1), anti-CD45 (clone F10-89-4) and anti-CD163 (clone GHI/61).

Western blot

Western blot was carried out utilizing the conventional SDS-PAGE separation method. The cells were rinsed twice with a pre-cooled PBS solution, and then lysed in RIPA buffer containing protease inhibitors on ice for 30 min. A 10% SDS-PAGE gel was used for electrophoresis, followed by blotting onto a PVDF membrane. After blocking with 5% milk for an hour, the membrane was incubated with the first antibody (Abcam, USA) overnight at 4 °C in a shaker, and then with the HRP-conjugated goat anti-mouse IgG (secondary antibody) at room temperature for 1 h with three TBST washes in between. The immunoreactive protein bands were visualized in a chemiluminescence instrument.

Transfection

THP-1 cells were seeded in 6-well plates (5 × 105/well) and cultured overnight to reach the confluency of ~ 80% before the transfections. siRNA-lipofectamine 2000 (Invitrogen, USA)mix were prepared according to the manufacturer’s instruction. After the addition of siRNA-lipofectamine 2000 mix, cells were incubated for a further 24 h before analysis.

Transwell assays

Transwells were inserted into the 24-well culture plates. Th1 cells (2 × 104 cells/ml) were placed in the upper chamber of the Transwell insert, while THP-1-derived macrophages, pre-treated with IFN-γ (1 ng/ml) for 24 h to induce CXCL10 productions, were seeded in the lower compartment. In some cases, STAT1-targeting siRNA was added into the low chamber. After 3 h, the number and morphology of migrated Th1 cells was determined by smear staining.

Statistical analysis

All statistical analyses were performed with the software SPSS 21.0. Count data were expressed as percentages or rates; normally distributed measures were expressed as mean ± standard deviation (SD), and Pearson’s correlation analysis was conducted. Data with skewed distribution were described as median (interquartile range) [M (P25, P75)], and Spearman’s correlation analysis was performed. P ≤ 0.05 was regarded as statistically significant.

Ethics statement

The study involving human participants complied with the Declaration of Helsinki and was reviewed and approved by the Institutional Human Ethics Committee of the Children’s Hospital of Soochow University under approval number 2023CS231. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.

Results

Demographic data, clinical and laboratory characteristics

A total of 122 children in the MPP group and 54 controls were enrolled in the present study. The demographic data, clinical and laboratory characteristics of children are shown in Table 1. In the MPP group, the mean age of the children was 6.86 ± 0.25 years, with 63/59 males/females accounting for 51.6%/48.4%, respectively. 11 patients (9.01%) had pleural effusion, and 29 (23.77%) had lobar pneumonia. Clinically, children with pneumonia mainly manifested with cough and fever, with an average fever duration of 7.6 ± 0.38 days and fever peak of 39 ± 0.34℃. The mean age of the control group was 6.37 ± 0.37 years, with 30 male (55.5%) and 24 female (44.5%).

Table 1 Demographic data and clinical characteristics of patients enrolled in this study.
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Altered CXCL10 protein levels in children with MPP

MPP can be classified into RMPP and NRMPP based on the clinical symptoms of the disease. RMPP is one of the complicated manifestations of MPP. RMPP is characterized by prolonged fever and deterioration of clinical and radiological findings, acute respiratory distress syndrome, and even necrotizing pneumonitis, regardless of appropriate antibiotic therapy. We first performed a protein chip analysis by using 3 blood samples from the groups of RMPP, NRMPP, and healthy patients to screen for differentially expressed proteins (DEPs) among these groups. Our data revealed that CXCL10 was the most significantly upregulated proteins in MPPs (P < 0.05; Fig. 1a). Moreover, ontology analysis showed that most DEPs related to the cytokine-cytokine receptor interaction pathway, such as cell migration/chemotaxis, chemokine-mediated signaling pathway and CXCR3 chemokine receptor binding (Fig. 1b). Then we used KEGG analysis22,23,24 to predict which the JAK/STAT signaling pathway would involve CXCL10 in the pathogenesis of NRMPP or RMPP (Fig. 1c).

Fig. 1
figure 1

CXCL10 protein expression exhibited the greatest difference between MPP patients and controls. (a) Heatmap showing the differentially expressed proteins (DEPs) in refractory M. pneumoniae pneumonia (RMPP) vs. Control ( left) and non-refractory M. pneumoniae pneumonia (NRMPP) vs. Control (right). (b) Ontology analysis of DEPs in RMPP vs. Control (left) and NRMPP vs. Control (right), with Pillar length denoting protein counts (BP biological process; CC cellular component; MF molecular function). (c) Ontology analysis of DEPs in RMPP vs. Control (left) and NRMPP vs. Control (right) in the cytokine-cytokine receptor interaction pathway of KEGG, with Pillar length denoting protein counts.

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Clinical significance of IFN-γ/CXCL10 in the peripheral blood and BALF

In line with data obtained in protein chip analysis, the expression levels of inflammatory factors CXCL10 and IFN-γ were significantly elevated in the peripheral blood and BALF of MMP patients than those in controls (Fig. 2a). Moreover, levels of CXCL10 were significantly higher in the BALF as compared to that in peripheral blood of MPP patients (Fig. 2b). Notably, blood CXCL10 levels were positively correlated with days of hospitalization (r = 0.565, P < 0.05), fever duration (r = 0.341, P < 0.05) and serum IFN-γ levels (r = 0.576, P < 0.0001), and BALF CXCL10 levels were positively correlated with total IgM in blood (r = 0.365, P < 0.05) in the MPP group (Fig. 2c). In addition, the numbers of neutrophils and macrophages in the alveolar lavage fluid of MPP patients were remarkably increased as compared to those in the control group, and the majority (> 80%) of macrophages in BALF of MPP patients exhibited a M1-polarized phenotype (Fig. 2d,e). In line with its biological function, CXCL10 levels showed a significant correlation with neutrophil/macrophages numbers in BALF (Fig. 2f).

Fig. 2
figure 2

Clinical significance of IFN-γ/CXCL10 in peripheral blood and BALF of children with MPP infections. (a) Protein levels of IFN-γ and CXCL10 in blood/BALF of MPP patients vs. controls. (b) Protein levels of CXCL10 in the blood vs. BALF of MPP patients. (c) Correlations between blood/BALF CXCL10 with duration of fever, hospitalization, serum IFN-γ or total IgM levels in patients with MPP. (d) Representative FACS-plots showing the phenotype of macrophages in BALF of MPP patients vs. controls. (e) Significantly increased numbers of neutrophils and macrophages, but not lymphocytes, in BALF of MPP patients. (f) Correlations between CXCL10 levels with neutrophil/macrophage numbers in BALF.

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STAT1 down-regulation inhibits IFN-γ-induced CXCL10 expression in M1-type monocytic macrophages

IFN-γ promoted the mRNA and protein levels of CXCL10 in THP-1-derived macrophages in a dose-dependent fashion in all time points (3, 6, and 9 h) analyzed (P < 0.05, Fig. 3a,b), accompanied with the increased phosphorylation of STAT1 (Fig. 3c), indicating that IFN-γ induces CXCL10 via STAT1. Indeed, siRNA-mediated downregulation of STAT1 significantly diminished the ability of activated Th1 cells (Fig. 3d) or IFN-γ (Fig. 3e) to elevate CXCL10 expressions in THP-1-derived macrophages as determined by western blot (Supplementary Fig. 1, Fig. 2 and Fig. 3), qPCR and ELISA. Moreover, si-RNA mediated downregulation of STAT1 repressed the ability of THP-1-derived macrophages to activate Th1 cells, as evidenced by the significantly reduced IFN-γ levels in culture supernatants (Fig. 3f).

Fig. 3
figure 3

IFN-γ induced CXCL10 expression in THP-1-derived macrophages in a STAT1-dependent manner. Macrophages, derived from PMA-stimulated THP-1 cells, were stimulated with different concentrations of IFN-γ (1 or 10 ng/ml) for indicated time (3,6, and 9 h) before the collection of cells and supernatants (a–c). (a,b) Levels of CXCL10 in THP-1-derived macrophages or culture supernatants were detected by qPCR (a) or ELISA (b). (c) Levels of CXCL10, STAT1 and phosphorylated STAT1 (p-STAT1) were analyzed by WB. (d) Levels of CXCL10, STAT1 and p-STAT1 were analyzed by WB, THP-1-derived macrophages were transfected with either control siRNA (si-Control) or STAT1-targeting siRNA (si-STAT1) before being cocultured with activated CD4 + Th1 cells. After 9 h of coculture, cells were harvested for further analysis. (e) mRNA and protein levels of CXCL10 was analyzed by qPCR/WB/ELISA in THP-1-derived macrophages/culture supernatants without (si-Control)/with knocking down of STAT1 (si-STAT1). Cells were activated with INF-γ for 9 h. (f) Expressions of INF-γ and CXCL10 were analyzed by ELISA and or qPCR. THP-1-derived macrophages were treated with control (si-Control) or STAT1-targeting (si-STAT1) siRNA before coculture with activated CD4+ Th1 cells. Cells and supernatants were collected after 9 h of coculture (The uncropped original blot/gel is shown in Supplementary Fig. 1, Fig. 2 and Fig. 3).

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CXCL10 promotes the migration of Th1 cells

The results we showed above indicated that IFN-γ and/or activated Th1 cells promotes CXCL10 productions in macrophages via STAT1. To determine the reciprocal effect of STAT1-induced CXCL10 in macrophages on Thcells, we next conducted transwell experiments. The addition of IFN-γ in the lower chamber greatly promoted the migration of Th1 cells, which was significantly diminished by siRNA-mediated knockdown of STAT1 (Fig. 4), indicating that macrophage-derived CXCL10, triggered by the IFN-γ/STAT1 axis, induces the transmigration of Th1 cells.

Fig. 4
figure 4

IFN-γ stimulated macrophages greatly promoted the migration of Th1 cells. THP-1-derived macrophages, pretreated with control (si-Control) or STAT1-targeting siRNA (si-STAT1), were seeded on the lower chamber while activated Th1 cells were placed in the upper insert of the transwell system. IFN-γ(1 ng/ml) was added to elicit CXCL10 productions by macrophages (blue and green bars). Numbers of transmigrated Th1 cells in the lower compartment were visualized by smear stainings. Scale bar in b = 50μm.

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Together, our data indicate that IFN-γ promotes the production of CXCL10 in macrophages via STAT1. Subsequently, CXCL10 recruits more IFN-γ-producing Th1 cells into the inflammatory sites, thereby constituting a vicious circle that exacerbates proinflammatory immune responses and tissue damages in patients with MPP infections.

Discussion

Mycoplasma pneumoniae is one of the primary pathogens that cause chronic respiratory diseases in humans. In recent years, the incidence of MPP has been rising, and in some cases, it has even become a serious or fatal condition in children25. The pathogenesis of MPP is complicated and can be classified into two main categories: extrapulmonary and intrapulmonary modes of infection. Intrapulmonary infections may relate to adhesion proteins, nutrient depletion, toxins, oxidative damage, inflammatory damage, and immune evasion. By contrast, extrapulmonary infections seem to mainly manifest as immune-mediated direct injury caused by inflammatory cytokines, or vascular occlusion as a result of thrombosis/vasculitis formation26,27. Upon MPP infection, the opsonization effect of antibodies/complements may induce a cytokine storm by activating various immune cells, including neutrophils, macrophages and lymphocytes, and thereby leads to serious tissue damages in the lungs. Thus, studies on the inflammatory responses induced by Mycoplasma pneumoniae as well as the underlying molecular mechanisms will benefit the diagnosis and treatment of MPP patients clinically.

We herein found that the expression levels of CXCL10 were significantly elevated in both the peripheral blood and BALF of MPP patients as compared to those in controls. Moreover, levels of CXCL10 were higher in BALF than that in the peripheral blood of patients with MPP, pointing to an ongoing local production at infection sites. Indeed, CXCL10 was positively correlated with clinical manifestations and laboratory indexes, including days of hospitalization, fever duration, as well as serum IFN-γ and IgM levels. These findings are consistent with previous studies showing that elevated levels of CXCL10 were linked to disease duration, progression, and severity in patients with MPP or COVID-2019 28,29,30. As such, CXCL10 has been used as a clinic maker for the diagnosis and/or prognosis of various disorders.

It has been shown that the levels of cytokines in blood samples from children, including IL-4, IFN-γ, IL-17, TNF-α, and CXCL10, were significantly increased in cases of MPP patients as compared to those in the control group31,32,33. Our study confirms and expands these findings by showing the significantly increased numbers of neutrophils and M1-type macrophages in the BALF of kids correlate to CXCL10 levels in BALF. It is thus conceivable that high local CXCL10 levels contribute, at least partially, to the recruitment of immune cells into BALF of patients with MPP. Despite the increase of both IL-4 (a signature cytokine of Th2 cells) and IFN-γ (a hallmark cytokine of Th1 cells) in MPP patients, the M1-phenotype dominated macrophages in BALF suggest the Th1-type immune response prevails in respiratory tracts/lungs of MPP patients. In support of this notion, it has been shown that IFN-γ induces CXCL10 expressions in a variety of cell types, including monocytes, endothelial cells and dendritic cells in autoimmune diseases. Upon binding to CXCR3, CXCL10 augments the migration/infiltration of CD8+ and Th1-type CD4+ effector T cells into the infection/inflammatory tissues34,35. Given that levels of CXCL10 are positive correlated with IFN-γ levels or with leukocytes numbers in BALF of MPP patients, and that IFN-γ stimulates M1-type macrophages to produce CXCL10, which subsequently promotes the migration of Th1 cells, we speculate that the reciprocal regulation of macrophages and Th1 cells, partially mediated via IFN-γ and CXCL10, amplifies the type 1 immune responses and thereby aggravates tissue damages in respiratory tracts of patients with MPP.

The JAK-STAT1 signaling pathway participates multiple cytokine signalings and thus is important for our body’s immune responses33. Despite that inflammatory responses in mouse lung tissues were attenuated by repression of the JAK2/STAT1-STAT3 signaling15, the role of JAK-STAT1 signaling pathway in human MPP infections has not been thoroughly studied. Our data presented here showed that siRNA-mediated downregulation of STAT1 not only significantly diminished IFN-γ induced CXCL10 production in macrophages, but also hampered the ability of macrophages to promote IFN-γ production and migration of cocultured CD4+ Th1 cells. Thus, the IFN-γ/STAT1/CXCL10 axis seems to dictate the reciprocal regulation between macrophages and Th1 cells, which forms a positive feedback loop to enhance both the numbers and the function of immune cells in inflammatory sites. Hence, the IFN-γ/STAT1/CXCL10 axis may represent a potential target for treatment of MPP patients.

In conclusion, we confirmed previous studies showing that CXCL10 expression is significantly elevated in MPP patients’ blood samples. Moreover, we demonstrated this finding in bronchoalveolar lavage fluid, where CXCL10 levels were positively correlate with IFN-γ, leukocyte numbers in BLAFs and some clinical parameters. Additionally, we further explored and demonstrated in vitro that IFN-γ/STAT1/CXCL10 participates in the positive feedback loop that amplifies the inflammatory responses and exacerbates tissue damages in respiratory tracts of children with MPP infections.

Limitations

Although this study provides valuable insights into the IFN-γ/STAT1/CXCL10 axis and its potential role in MPP pathogenesis, some limitations remain, particularly the lack of in vitro and in vivo validation using MPP-specific models. Therefore, further refinement is required in future studies.