This study included patients who were diagnosed with MPP from July 2019 to October 2021 at Children’s Hospital of Soochow University (N=140); all diagnoses were confirmed by both MP-immunoglobulin (IgM) positivity and the presence of MP-DNA (≥1.0×10⁴ copies/ml) in nasopharyngeal aspirates(NPA) or BALF, as measured by real-time quantitative polymerase chain reaction (qPCR) (19). The patients’ ages ranged from 17 months to 16 years; their clinical manifestations included fever, cough, tachypnea, chest retractions, abnormal auscultatory findings, and radiologic evidence of CAP.

Among MPP patients, 117 cases were selected as experimental group A to study the correlation of CXCL9 expression level in peripheral blood; while 23 cases were selected as experimental group B and measured IFN-γ and CXCL9 in peripheral blood and BALF; to further study the correlation between IFN-γ/CXCL9 and immune inflammatory damage in the lung.

Cases met one or more of the exclusion criteria were excluded (a): co-infection (b); clinical and imaging features indicative of fungal pneumonia (c); presence of congenital respiratory diseases, and abnormalities in other systems (e.g., heart, liver, kidney, and blood) (d); >3 days of treatment with glucocorticoids (20) and/or azithromycin before admission. To exclude co-infections, seven respiratory virus antigen tests (influenza A and B; parainfluenza 1, 2, and 3; respiratory syncytial virus; and adenovirus) and other pathogen tests (human bocavirus; human rhinovirus; human metapneumovirus; and chlamydia pneumoniae) were conducted; the results were negative. The results of bacterial cultures of NPA and BALF were also negative.

Peripheral blood and BALF were also collected from 18 children who underwent emergency surgery for foreign bodies in the ENT department of our hospital during the same period with 23 cases of MPP children; these children served as a control group. The inclusion criteria were as follows: no respiratory tract infection within the previous 4 weeks, no chronic lung disease or bronchopulmonary malformation, and no history of treatment with hormones or immunosuppressive agents.

The studies involving human participants were reviewed and approved by the Ethics Committee of the Children’s Hospital of Soochow University (2019LW014) on July 24, 2019. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. All participant data were anonymized prior to analysis.

All patients’ age and sex were recorded. In the experimental group A, the following clinical data were recorded: duration of fever, fever peak, and imaging manifestations. Peripheral blood samples obtained within 24 h of admission were used for measurements of CXCL9 and specific antibodies to M. pneumoniae.

In the experimental group B, the following clinical data were also recorded: durations of hospital stay and fever, fever peak, and laboratory test data on admission (Table 1). Peripheral blood samples obtained within 24 h of admission were also used for measurements of complete blood counts, C-reactive protein (CRP), lactate dehydrogenase (LDH), immunoglobulins (IgA/IgG/IgM), lymphocyte subsets, specific antibodies to M. pneumoniae, IFN-γ and CXCL9. Flexible fiber bronchoscopy and bronchoalveolar lavage were performed in accordance with existing guidelines (21). BALF was gently aspirated, collected, and prepared for detection of the protein concentrations of IFN-γ and CXCL9.

Nucleic acid extraction and qPCR for the detection of M. pneumoniae 16S rDNA were performed as previously described (17, 21). Briefly, samples (NPA/BALF) were shaken for 30s, then centrifuged at 12,000×g for 5 min. Subsequently, the sediment was collected and DNA was extracted from 400-μL of each sample. The primers for M. pneumoniae 16S rDNA were as follows: forward: 5′-GCAAGGGTTCGTTATTTG-3′; reverse: 5′-CGCCTGCGCTTGCTTTAC-3′ (amplicon size: 380 bp).

Real-time qPCR was performed using the iQ5TM BIO-icycler (Bio-Rad, Hercules, CA, USA). The PCR conditions were as follows: 37°C for 2 min; initial denaturation at 94°C for 10 min, followed by 40 cycles of denaturation at 94°C for 10s, annealing at 55°C for 30s, and extension at 72°C for 40s. After amplification, a computer connected to the instrument automatically analyzed the results, then expressed the test results as Ct values. For samples with a Ct value <38, a quantitative reagent (Aikang Co., Ltd., Hangzhou, China) was used for further quantitative determination.

IFN-γ and CXCL9 were measured in peripheral blood or/and BALF samples from MPP and control groups using the appropriate commercial enzyme-linked immunosorbent assay kits, in accordance with the manufacturer’s instructions. Enzyme-linked immunosorbent assay kits were purchased from Xuguang kexing Biotechnology Co., Ltd. (Suzhou, China).

Flow cytometry was used to determine cell counts of Mφs, and lymphocytes in BALF from children with MPP, using flow cytometry equipment (Beckman Coulter, Miami, FL). The forward scatter (FSC) value represents cell volume; a larger FSC value indicated greater cell volume. The side scatter (SSC) value represents cell granularity; a larger SSC value indicated higher granularity. Cells with greater volume and higher granularity were considered neutrophils; cells with less volume and lower granularity were considered lymphocytes; monocytes (Mφs) were in between the two types of cells; then, Mφ phenotypes were determined by flow cytometry.

Protein microarrays enable determination of the protein-binding specificities of multiple analytes in solution; they are versatile tools for high-throughput analyses of the human proteome (22). In a preliminary experiment, six blood samples from the SMPP group, four blood samples from the MPP group, and five blood samples from the control group were analyzed by Ray Biotech Co., Ltd. (Guangzhou, China), which provided all reagents and instruments.

Multiple protein molecules were immobilized on a solid support in a predetermined arrangement to form a microarray. Samples were incubated with the protein chip; after specific binding reactions occurred, the components that were not bound to the protein on the chip were removed by washing. Fluorescently labeled antibodies were used for secondary incubation; the fluorescence value of each point on the chip was analyzed using a commercial fluorescence scanning analysis instrument and software.

The animal study was reviewed and approved by the Ethics Committee of Soochow University (SUDA20200510A02) on May 10, 2020. C57BL/6 mice (age, 4–5 weeks; weight, 20±2g) were purchased from the Laboratory Animal Center of Soochow University (Suzhou, China). All animals were housed in separate cages under constant temperature (25±1°C) and humidity (50%) with 12-h light-dark cycles; they had free access to food and water.

Bone marrow mononuclear cells (BM-MNCs) were isolated from C57BL/6 mouse bone marrow via density gradient centrifugation. BM-MNCs were cultured in 24-well plates with Mouse Steady-State Dendritic Cell Culture Kit (Dongling Biotechnology Co., Ltd., Suzhou, China); the cell density was adjusted to 1.5×10⁶ cells/ml. Flt3L (Jinsirui Biotechnology Co., Ltd., Nanjing, China) was added to each well at a final concentration of 200 ng/ml; we obtained suspension cultured cells after 9 days of incubation (23), which were DCs with diverse phenotypic characteristics (i.e., F-DCs). Cultures were incubated at 37°C in a humidified atmosphere containing 5% CO₂ in air.

Recombinant CARDS toxin was constructed from Sino Biological Co., Ltd. (Beijing, China) and its biological function was verified as previously described (17, 21). According to the manufacturer’s instructions, the recombinant CARDS toxin was expressed in insect cells, and the insect cell expression system belonged to the eukaryotic cell expression system. From the NCBI database, we obtained the full-length gene sequence and protein sequence of MPN372, which coded CARDS Toxin. The optimized MPN372 gene sequence was cloned into the pFastBac donor plasmid vector for virus packaging. High-Five cells in logarithmic growth phase were infected with high titer recombinant virus to express the target protein, which was further purified by nickel column.

CD4⁺ T cells (1×10⁵ cells/ml) were isolated from mouse spleens in accordance with the instructions of the Mouse Naïve CD4⁺ T Cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada). Labeled cells were isolated using EasySep magnets without a column, and the target cells were transferred to new tubes. Naïve CD4⁺ T cells from the spleen were negatively selected using magnetic beads. Flow cytometry equipment was described as previously.

F-DCs were screened for phenotype by flow cytometry and the cell concentration was adjusted to 1×10⁵ cells/well. F-DCs incubated with cell growth medium were used as the negative control group; F-DCs incubated with CARDS toxin (10 ng/ml) were used as the experimental group. Both groups of cells were co-incubated with naïve CD4⁺ T cells (1:4 ratio of DCs to T cells) for 24 h at 37°C with 5% CO₂. The numbers of CD4⁺ IFN-γ⁺ Th, CD4⁺ IL-4⁺ Th, and CD4⁺ IL-17⁺ Th cells were analyzed by flow cytometry.

THP-1 cells in suspension (1×10⁵ cells/ml) were purchased from Noble Biological Co., Ltd. (Shanghai, China). Phorbol ester (PMA) was added to the THP-1 cells in suspension at a final concentration of 50 ng/ml. Then, these cells were seeded into six-well plates (1×10⁵ cells/ml), and adherent mononuclear Mφs were obtained after incubation for 24 h. The cell growth medium was then changed to complete medium without PMA; cells were cultured for 3 days to observe changes in morphology. Pseudopodia were observed, indicating successful Mφs induction.

IFN-γ has been reported to induce Mφs differentiation into M1-type Mφs (15). Here, we stimulated Mφs (1×10⁵ cells/ml) with different concentrations of IFN-γ (0 ng/ml, 1 ng/ml, or 10 ng/ml) for 24 h; we used two experimental methods (i.e., real-time qPCR and ELISA), to measure the CXCL9 expression level at 3 h, 6 h, and 9 h. The primers were synthesized by Jinkairui Biological Engineering Co., Ltd. (Wuhan, China). ELISA kits were purchased from ELK Biotechnology Co., Ltd. (Wuhan, China). Primer sequences are shown in Supplementary Table S1.

The Western blot is an important laboratory technique that allows for specific identification and characterization of proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins are electophoretically transferred to a polyvinylidene fluoride (PVDF) membrane which is then incubated with specific antibodies, then developed to show the protein of interest; β-actin is acted as an internal control. We determined the expression of protein CXCL9, STAT1 and p-STAT1 by Western Blot at different concentrations (IFN-γ 0 ng/ml, 1 ng/ml, or 10 ng/ml) and different times (3 h, 6 h, and 9 h). Reagents were purchased from ASPEN Biotechnology Co., Ltd. (Wuhan, China).

STAT1-siRNA-CON (negative control group), STAT1-siRNA-1, STAT1-siRNA-2, or STAT1-siRNA-3 were transfected into Mφs (1×10⁵ cells/ml) using Lipofectamine^® 2000 (Thermo Fisher Scientific, Waltham, MA, USA); the cells were incubated for 24 h after transfection. The effect of STAT1 interference (STAT1-siRNA-1, STAT1-siRNA-2, and STAT1-siRNA-3) on the expression of STAT1 in Mφs was analyzed by qPCR. The results of STAT1-siRNA interference efficiency verification showed that the three pairs of STAT1-siRNA down-regulated the expression level of STAT1 by 43%, 70%, and 80%, respectively; it met the quality control standards (at least one pair of siRNAs had a silencing efficiency of more than 70% at the mRNA level under standard conditions of use).

IFN-γ (1 ng/ml) was used to stimulate Mφs that had been transfected with STAT1-siRNA-1; after IFN-γ stimulation for 24 h, the level of CXCL9 expression was analyzed by qPCR and ELISA; CXCL9, STAT1 and p-STAT1 protein expression were analyzed by Western blot.

CD4⁺ T cells (1×10⁵ cells/ml) were isolated from the peripheral blood of healthy volunteers using autoMACS columns with the Direct Human CD4⁺ T Cell Isolation Kit (STEMCELL Technologies). CD4⁺ T cells were treated with PMA (20 ng/ml) + ionomycin (1 μg/ml) at 37°C for 1 h; Breedellin A (1 μl/ml) was then added and cells were cultured for an additional 3–5 h to obtain Th1 cells.

In the lower chamber of the Transwell plate (Corning, NY, USA), Mφs (1×10⁵ cells/ml) were stimulated with IFN-γ (1 ng/ml) for 24 h; one group was transfected with STAT1-siRNA-1, whereas one group was not transfected. Unstimulated Mφs were used as negative controls. In the upper chamber of the Transwell plate, Th1 cells were seeded. The suspension in the lower chamber was subjected to smear staining to analyze the cell morphology and determine the number of migrating Th1 cells.

Data were stored in a Microsoft Excel-supported database, statistical analyses were performed using SPSS statistics version 20.0 (International Business Machines Corp., NY), and graphics were prepared using GraphPad Prism version 6.0 (GraphPad Software Inc., San Diego, CA). Categorical data of patient characteristics were compared using the Chi-square test. Data with normal distributions were expressed as means ± standard deviations; comparisons among groups were performed by t-tests or one-way analysis of variance. Data with non-normal distributions were expressed as medians (interquartile ranges); comparisons between groups were performed by the Mann–Whitney U-test. The Spearman correlation coefficient was used for correlation analysis. P-values <0.05 were considered statistically significant.

Demographic data and clinical characteristics were collected uniformly from the control and MPP groups (experimental group A and experimental group B). In experimental group A, there were 52 (44.44%) male and 65 (55.56%) female cases, with a male to female ratio of 1:1.25 and a mean age of 5.74 ± 3.03 years.

In experimental group B, there were 8 (34.78%) male and 15 (65.22%) female cases, with a male to female ratio of 1:2.25, while in the control group there were 10 (55.56%) male and 8 (44.44%) female cases, with a male to female ratio of 1.25:1, as shown in Table 1. There was no significant difference in terms of sex between the control group and experimental group B (P>0.05) and there was a difference in terms of age between two groups (P<0.05). Most airway foreign bodies in children occur in children under 3 years old and the peak incidence is in children aged 1-2 years. Therefore, specimens will to be collected to verify the difference level of IFN-γ/CXCL9 between the two groups in the further. The duration of fever was 6.78 ± 2.76 days, the fever peak was 39.36 ± 0.59°C, and the duration of hospitalization was 9.13 ± 2.26 days, as shown in Table 1.

To identify differentially expressed proteins (DEPs) in patients with M. pneumoniae infection, we selected six blood samples from the SMPP group, four blood samples from the MPP group, and five blood samples from the control group for protein microarray analysis in a preliminary experiment. DEPs were defined as proteins with P<0.05 and fold change >1.2 or <0.83 (absolute log₂ [fold change] >0.263).

In terms of DEPs with significant expression, the levels of CXCL9 (MIG), CXCL10 (IP-10), and IL-18 changed between the two groups (Figures 1A, D). Gene Ontology (GO) enrichment analysis between the two groups was performed at three levels: cellular component, molecular function, and biological process. DEPs were related to CXCR3 chemokine receptor binding, cytokine response, and cell chemotaxis and migration (Figure 1B). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that DEPs might be related to the JAK/STAT signaling pathway (Figure 1C). Further clinical pre-experimental research was conducted in the early stage through detecting the same samples by ELISA; only the level of CXCL9 expression significantly differed between the two groups (P<0.05) (Figure 1E). The level of IFN-γ expression is reportedly closely associated with MPP severity and subsequent recovery; moreover, IFN-γ can induce the production of CXCL9 (24). The above findings suggested that CXCL9 plays an important role in M. pneumoniae infection.

In experimental group A, the duration of fever was 4.52 ± 2.96 days, the fever peak was 39.05(38.70, 39.70) °C. The CXCL9 expression level was upregulated among patients with a higher fever peak, fever duration of greater than 7 days, an imaging manifestation of lobar or segmental, or combined pleural effusion (All P<0.05, Figure 2).

In experimental group B, the peripheral blood levels of IFN-γ and CXCL9, which were higher in patients than in the healthy control group, were positively correlated with each other (r=0.502, P<0.05). In patients, the CXCL9 expression level was significantly higher in the BALF than in the peripheral blood, and the BALF CXCL9 expression level was higher than that in the healthy control group (All P<0.05, Figure 3A). The expression level of IFN-γ in BALF between experimental group B and control group was low.

CXCL9 in peripheral blood was negatively correlated with the duration of fever, whereas CXCL9 in BALF was positively correlated with the duration of fever (r=-0.43 and r=0.44, respectively; both P<0.05). CXCL9 in peripheral blood was positively correlated with neutrophils(N)%, whereas CXCL9 in BALF was positively correlated with eosinophils(EOS)% (r=0.54 and r=0.42, respectively; both P<0.05; Figure 3B). Other laboratory test data showed no significant correlations, as shown in Table 1.

We selected 5 children with MPP in experimental group B and 5 children in the control group to measure cell counts and Mφ phenotypes in BALF by flow cytometry. Results showed that the cell counts of Mφs were significantly higher than those of the control group (t=2.844, P<0.05; Figure 3C). Previous studies have shown that Mφs mainly comprise two distinct functional phenotypes: classically activated Mφs (M1) and alternatively activated Mφs (M2) (25). Our flow cytometry analysis revealed that M1-phenotype Mφs (CD16⁺ CD64⁺ CD163^- ) were predominant (Figure 3D).

Mouse BM-MNCs were isolated, then differentiated into F-DCs using Flt3L; CD11c⁺ DCs were identified by flow cytometry (Figure 4A). CD4⁺ T cells with low carboxyfluorescein diacetate succinimidyl ester (CFSE) staining were promoted after CARDS toxin stimulation (P<0.001, Figure 4B). Notably, F-DCs stimulated with CARDS toxin promoted the differentiation of CD4⁺ IFN-γ⁺ Th (Th1) cells(P<0.001), without affecting the differentiation of Th2 or Th17 cells (Figure 4C).

IFN-γ promoted the level of CXCL9 expression in a dose-dependent manner at the same time (3 h, 6 h, and 9 h) by qPCR and ELISA (P<0.05); qPCR and ELISA also showed that the level of CXCL9 expression significantly increased after stimulation with IFN-γ (1 ng/ml, 10 ng/ml) (P<0.05). Upon stimulation with the same concentration of IFN-γ(1 ng/ml), the level of CXCL9 expression significantly decreased after Mφs had been transfected with STAT1-siRNA-1 (P<0.05) (Figures 5A, C, D).

Western Blot showed protein CXCL9 and p-STAT1 were expressed in a dose-dependent and time-dependent manner (P<0.05). The expression of protein CXCL9, STAT1 and p-STAT1 increased after stimulation with IFN-γ (1 ng/ml) (P<0.05); after Mφs had been transfected with STAT1-siRNA-1, the expression of these proteins significantly decreased (P<0.05) (Figures 5B, E).

The expression of CXCL9 in the lower Transwell chamber increased after 24 h of Mφ (THP-1+PMA induction) stimulation with IFN-γ (1 ng/ml), and the number of migrating Th1 cells increased (P<0.001). Whereas, the number of migrating Th1 cells significantly decreased after Mφs had been transfected with STAT1-siRNA-1 (P<0.001) (Figure 5F).

An important limitation in this study was that we did not characterize the specific mechanism by which CARDS toxin stimulates F-DCs and promotes Th1 cell differentiation. We will continue to focus on this mechanism in future studies.