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An anti-complement homogeneous polysaccharide from Houttuynia cordata ameliorates acute pneumonia with H1N1 and MRSA coinfection through rectifying Treg/Th17 imbalance in the gut–lung axis and NLRP3 inflammasome activation
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Xinxing Lia, Wenxin Dingb, Yan Lua, Haiyan Zhub, Weilian Baoa, Yang Liua, Jiaren Lyua, Lishuang Zhoua, Hong Lic, *, Jiyang Lib, *, Daofeng Chena, *
Acta Pharmaceutica Sinica B | 2025, 15(6) : 3073 - 3091
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Acta Pharmaceutica Sinica B | 2025, 15(6): 3073-3091
• ORIGINAL ARTICLE •
An anti-complement homogeneous polysaccharide from Houttuynia cordata ameliorates acute pneumonia with H1N1 and MRSA coinfection through rectifying Treg/Th17 imbalance in the gut–lung axis and NLRP3 inflammasome activation
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Xinxing Lia, Wenxin Dingb, Yan Lua, Haiyan Zhub, Weilian Baoa, Yang Liua, Jiaren Lyua, Lishuang Zhoua, Hong Lic, *, Jiyang Lib, *, Daofeng Chena, *
Affiliations
  • aDepartment of Natural Medicine, School of Pharmacy, Fudan University, Shanghai 201203, China
  • bDepartment of Biological Medicines, School of Pharmacy, Fudan University, Shanghai 201203, China
  • cDepartment of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, China
doi: 10.1016/j.apsb.2025.04.008
Outline
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The coinfection of respiratory viruses and bacteria is a major cause of morbidity and mortality worldwide, despite the development of vaccines and powerful antibiotics. As a macromolecule that is difficult to absorb in the gastrointestinal tract, a homogeneous polysaccharide from Houttuynia cordata (HCPM) has been reported to exhibit anti-complement properties and alleviate influenza A virus (H1N1)-induced lung injury; however, the effects of HCPM without in vitro antiviral and antibacterial activities on more complicated pulmonary diseases resulting from viral-bacterial coinfection remains unclear. This study established a representative coinfection murine pneumonia model infected with H1N1 (0.2 LD50) and methicillin-resistant Staphylococcus aureus (MRSA, 107 CFU). HCPM significantly improved survival rate and weight loss, and ameliorated gut–lung damage and inflammatory cytokine production. Interestingly, the therapeutic effect of HCPM on intestinal damage preceded that in the lungs. Mechanistically, HCPM inhibited the overactivation of the intestinal complement (C3a and C5a) and suppressed the activation of the NLR family pyrin domain-containing 3 (NLRP3) pathway, which contributes to the regulation of the Treg/Th17 cell balance in the gut–lung axis. The results indicate the beneficial effects of an anti-complement polysaccharide against viral–bacterial coinfection pneumonia by modulating crosstalk between multiple immune regulatory networks.

Houttuynia cordata  /  Polysaccharides  /  H1N1  /  MRSA  /  Pneumonia  /  Complement  /  NLRP3  /  Treg/Th17 cell balance  /  Gut-lung axis
Xinxing Li, Wenxin Ding, Yan Lu, Haiyan Zhu, Weilian Bao, Yang Liu, Jiaren Lyu, Lishuang Zhou, Hong Li, Jiyang Li, Daofeng Chen. An anti-complement homogeneous polysaccharide from Houttuynia cordata ameliorates acute pneumonia with H1N1 and MRSA coinfection through rectifying Treg/Th17 imbalance in the gut–lung axis and NLRP3 inflammasome activation[J]. Acta Pharmaceutica Sinica B, 2025 , 15 (6) : 3073 -3091 . DOI: 10.1016/j.apsb.2025.04.008
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1.　Introduction
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Pulmonary infections caused by viral and bacterial coinfection are serious diseases threatening human life. Studies have shown that coronavirus disease 2019 (COVID-19) patients with a bacterial coinfection or secondary infection have a higher in-hospital mortality and longer length of hospitalization1,2. Most influenza-related deaths occur from bacterial superinfections rather than the direct effects of the influenza virus3. During other respiratory viral infections, such as the Middle East respiratory syndrome and severe acute respiratory syndrome, the incidence of bacterial coinfection was reported as 1%–19%4-6 and 1%–43%4, respectively. Of note, methicillin-resistant Staphylococcus aureus (MRSA) is the most commonly encountered coinfections bacterium associated with multiple respiratory infections including the influenza pandemic and COVID-19. Currently, the clinical treatment for virus–bacterium coinfection pneumonia includes a combination of antiviral drugs and antibiotics. Coinfection of viruses and bacteria can quickly develop into severe, necrotizing pneumonia, causing over 50% mortality despite antibiotic treatment7. Antibiotics or antiviral drugs work directly against the pathogen. Respiratory coinfections involve complex interactions between viruses/bacteria and the host8,9. Therefore, targeting the host immune system might be the potential strategy for coinfection treatment without the risk of antiviral and/or antibiotic resistance8.
The complement system is an important component of the innate immune system and is vital for the host defense against microbial infections10,11. However, the inappropriate activation of the complement system is involved in the pathogenesis of multiple tissue injuries and acute inflammatory diseases, such as acute respiratory distress syndrome12-14. The complement activation products, anaphylatoxins C3a and C5a, activate the inflammatory response as well as fatal shock-like syndrome12,15. Anti-C5a antibody or anti-C3 agents (such as AMY-101) can lead to immediate clinical improvement in COVID-1916. Regulation of complement activity may be beneficial for the treatment of a wide range of complement-associated diseases16,17.
C3a and C5a induce the NLR family pyrin domain-containing 3 (NLRP3) inflammasome18-21. Activated NLRP3 binds to its adaptor, apoptosis-associated speck-like protein containing a CARD (ASC), which in turn, interacts with caspase-1 to form a complex known as the inflammasome, leading to caspase-1 activation. Caspase-1 subsequently cleaves the proinflammatory cytokines IL-1β and IL-18 into their active forms, thus contributing to an uncontrolled inflammatory response22,23. Many studies have shown that the NLRP3 inflammasome is an important molecular pathway that mediates the response against a myriad of pathogenic microbial infections24. However, NLRP3 appears to play a different role in mice during viral and bacterial pneumonia24. In NLRP3−/− mice, the survival rate during viral infection is decreased24. Bacterial infection of mice lacking NLRP3 results in decreased lung inflammation, which indicates its protective role25.
An imbalance between Treg and Th17 cells has been observed in the majority of pulmonary inflammatory diseases26-28. More importantly, our previous study has shown that the migration of Treg/Th17 cells in the gut-lung axis is affected by the chemokine CCR6 and its ligand CCL20, and confirmed that Treg/Th17 cells in the gut-associated lymphoid tissue (GALT) affect Treg/Th17 cells in the lung mucosa through CCR6–CCL20 axis26. Interestingly, multiple lines of evidence suggest that the NLRP3 inflammasome is involved in Treg/Th17 cell balance in multiple models of inflammation, including asthma23, arthritis29, and ulcerative colitis30. Nevertheless, the effects of complement, the NLRP3 inflammasome, and Treg/Th17 cells in the viral–bacterial coinfection murine model remain uncertain.
As a traditional Chinese medicine (TCM) that clears heat and eliminates toxins, the Houttuynia cordata Thunb. is commonly used for the treatment of infectious lung diseases, cancer, and anaphylaxis31-33. Polysaccharides from Houttuynia cordata, as macromolecular substances that are not easily absorbed into the blood, have attracted much attention because of their prominent immunomodulatory effects26. In our previous studies, crude polysaccharides from Houttuynia cordata (HCP) and/or a homogeneous polysaccharide from Houttuynia cordata (HCPM) ameliorated lung injury induced by influenza A virus (H1N1) or lipopolysaccharide. The primary mechanism was associated with the inhibition of the excessive activation of the complement system and inflammation11,34,35. The results indicated that anticomplementary polysaccharide may be a key active substance in HCP for treating pulmonary infection.
Crosstalk between the gut and lung, known as the “gut–lung axis”, is critical for the immune response in respiratory diseases36. Interestingly, the intestine has often been viewed as a potential target organ for polysaccharides37,38. Our previous studies indicate that HCP has significant anti-complement properties that alleviate H1N1-induced pneumonia by regulating the Treg/Th17 cell balance in the gut–lung axis26. However, whether this anticomplementary polysaccharide can modulate the complement system and T cell homeostasis and thus exert a beneficial effect in a virus and bacteria coinfection model, especially drug-resistant bacteria including MRSA, remains unclear. The specific mechanism of action of this polysaccharide is also unknown.
In this study, we used the H1N1–MRSA coinfection mouse model to examine the therapeutic effects and potential immunomodulatory mechanisms of HCPM on virus-bacterium coinfection-induced severe pneumonia. We evaluated the multitarget effects of HCPM in this coinfection model in terms of complement, NLRP3 inflammasome, and Treg/Th17 cell balance.
2.　Materials and methods
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2.1.　 Reagents and antibodies
PBS (#ST476), RIPA lysis buffer (#P0013B), phenylmethanesulfonyl fluoride solution (PMSF, #ST507), SDS-PAGE gel preparation kit (#P0012A), TBS with tween-20 (TBST, #ST673), Na, K-ATPase α1 rabbit monoclonal antibody (#AG1183), GAPDH rabbit monoclonal antibody (#AF1186), and HRP-labeled goat anti-rabbit IgG (H + L) (#A0208) were purchased from Beyotime (Shanghai, China). Xylene (#10023418) and absolute ethanol (#100092680) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Isoflurane (#20220801) was obtained from Jindafu Pharmaceutical Co., Ltd. (China). Mannitol salt agar (#HB4128) and nutrient broth (NB, #HB0108) were purchased from Qingdao Hope Biotechnology Company (China). Nonfat dry milk (#1706404) was purchased from Bio-Rad (Hercules, CA, USA). MCC950 (#M8083) was obtained from AbMole BioScience (Houston, TX, USA). ZO-1 (#21773-1-AP) antibody was purchased from Proteintech (Rosemont, IL, USA). Claudin-1 (#sc-166338) and Occludin (#sc-133256) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). NLRP3 (#T55651), Cleaved-caspase-1 (#TA4022), and Caspase-1 (#PA5426) were purchased from Abmart (Shanghai, China). ASC (#13833S) antibody was purchased from Cell Signaling Technologies (Beverly, MA, USA). Complement C3a (#144-60264) antibody was obtained from RayBiotech (Atlanta, GA, USA). Complement C5a (#PA5-78891) antibody was purchased from Thermo Fisher Scientific (Waltham, MA, USA). H1N1 NP (#NB100-56570) was purchased from Novus Biologicals (Littleton, CO, USA). The ELISA kits for mouse TNF-α (#EK282/4-96), mouse IL-6 (#EK206/3-96), mouse MPO (#EK2133/2-96), mouse IFN-γ (#EK280/3-96), mouse IL-10 (#EK210/4-96), mouse IL-17A (#EK217/2-96), mouse IL-18 (#EK218), and mouse IL-1β (#EK201B) were purchased from Multisciences Biotech Company (Hangzhou, China). Mouse IFN-α (#EMC035a) and mouse IFN-β (#EMC016) were purchased from Neobioscience (Shenzhen, China).
2.2.　 Animals, virus, and bacterial strain
Female mice were firstly selected to establish the H1N1 and MRSA coinfection model according to our previous experience in a mouse model of H1N1 infection. C57BL/6 female mice (14–16 g, four weeks old) were purchased from Slaccas Experimental Animal Inc. (Shanghai, China). The mice were housed in a specific-pathogen-free environment with a controlled temperature of 24 ± 2 ℃ and subjected to a 12-h light/dark cycle with free access to food and water. The animal experiments were approved by the Animal Ethics Committee of Fudan University (License number: 2020-09-SY-LJY-01) and carried out in compliance with the guidelines.
The mouse-adapted influenza A virus strain (A/FM/1/47, H1N1) was provided by Dr. Haiyan Zhu (School of Pharmacy, Fudan University). The half-lethal dose (LD50) for H1N1 virulence was calculated by the method of Reed-Muench39. The virus was re-packaged and stored at −80 ℃. MRSA-20-385 (Ethical number: 2019-460) was obtained from the Huashan Hospital Affiliated to Fudan University (Shanghai, China). The bacterial strains were stored at −80 ℃ in brain heart infusion broth supplemented with 20% glycerol. The bacteria were inoculated in 6 mL of nutrient broth (#HB0108, Qingdao Hope Bio-Technology Company) at a ratio of 1:100 and shaken horizontally at 180 rpm for 18 h at 37 ℃ in a constant temperature shaker (Jing Hong Laboratory Instrument Company, Shanghai, China). After two growth cycles, turbidity was calculated using a turbidity meter (#WZT-1M, Jinjia Scientific Instrument Company, Shanghai, China). The bacterial pellets were collected following centrifugation. The pellets were washed twice with sterile sodium chloride solution (#L222062507, Sichuan Kelun Pharmaceutical Company, Chengdu, China) and diluted to three experimental concentrations of 108, 107, and 106 colony-forming units (CFU) based on the turbidity 40.
2.3.　 Cellular toxicity of Houttuynia cordata polysaccharides on Madin–Darby canine kidney (MDCK) cells
Cellular toxicity was measured as described previously with minor modification41. Briefly, MDCK cells were seeded into 96-well plates (5 × 104 cells/well) and cultured for 14 h. The cells were then treated with HCPM (50, 100, and 200 μg/mL) or HCP (200 μg/mL) for 24 h. Toxicity was determined using an MTT assay kit (#C0009S, Beyotime, Shanghai, China) based on the manufacturer's instructions. The absorbance was measured with a microplate reader (Thermo Scientific, Waltham, MA, USA) at 570 nm.
2.4.　 In vitro anti-influenza virus H1N1 effect
An in vitro anti-H1N1 assay was conducted as described previously42. MDCK cells were seeded into 96-well plates (5 × 104 cells/well) overnight and infected with the H1N1 virus (2 × 10−4) for 2 h. The cells were then treated with HCPM (50, 100, and 200 μg/mL) or HCP (200 μg/mL) for 3 days. A cytopathic effect reduction assay was used to evaluate the antiviral effects of the test samples.
2.5.　 In vitro anti-MRSA assay
An in vitro anti-MRSA assay was performed as described previously43. Briefly, bacterial suspensions were added to a nutrient broth at a final concentration of 106 CFU/mL. The bacterial suspensions were treated with HCPM or HCP for 24 h. Finally, the OD600nm of each well was measured using a microplate reader to determine the concentration that inhibited MRSA growth.
2.6.　 Murine model of viral-bacterial coinfection
To construct a milder coinfection model consistent with the characteristics of TCM, we further explored the infective dose of MRSA based on 0.2 LD50 H1N144. C57BL/6 mice were infected intranasally with an extremely low dose (0.2 LD50) of H1N1, followed by MRSA (108, 107, or 106 CFU) infection three days later during light isoflurane anesthesia. These mice were euthanized on 5/6 days post H1N1 infection (dpi) and the changes in body weight and lung pathology were observed.
2.7.　 Treatment with Houttuynia cordata polysaccharides
The HCPM (Mw, 19.1 kDa) and HCP were prepared and identified by Dr. Lishuang Zhou (School of Pharmacy, Fudan University, Shanghai, China), and relevant chromatograms were reported in the study35 by Dr. Lishuang Zhou. The dose of HCPM and HCP was based on the results of the previous study45 and pilot tests. The mouse model in this study was established by coinfection with H1N1 and MRSA. Oseltamivir is a standard antiviral drug for H1N1 influenza virus. Linezolid is an antibiotic used clinically to treat MRSA infections. Although existing study46 has used Oseltamivir as a positive control for H1N1 and MRSA coinfections, which highlights the importance of antiviral therapy, the status of the bacteria is often neglected. Clinical guidelines47 suggest the early use of antiviral therapy for influenza patients with bacterial coinfections, followed by the administration of empirical antibiotics depending on the patient's condition. In this study, we designed three positive control groups: Oseltamivir, Linezolid, and Oseltamivir + Linezolid. This is consistent with the clinical guidelines and makes the experimental design more adequate.
Survival experiment: C57BL/6 mice were randomly divided into the following nine groups (11 mice/group): control, H1N1–MRSA coinfection (coinfection), coinfection + HCPM 20 mg/kg, coinfection + HCPM 40 mg/kg, coinfection + HCPM 80 mg/kg, coinfection + HCP 80 mg/kg, coinfection + Oseltamivir 22.75 mg/kg (coinfection + OST), coinfection + Linezolid 200 mg/kg (coinfection + LZD), and coinfection + Oseltamivir (22.75 mg/kg) + Linezolid (200 mg/kg) (coinfection + OST + LZD). Mice in the control group were intranasally administered with 30 μL of sterile sodium chloride solution without H1N1 or MRSA. The other mice were infected intranasally with 0.2 LD50 H1N1 (30 μL) and then infected with 107 CFU MRSA (30 μL) three days later. The mice in the control group were administered 0.5% sodium carboxymethyl cellulose solution (Aladdin, Shanghai, China) as the vehicle. The other mice were treated with the indicated drugs once daily for seven days via gavage, beginning 2 h after viral infection. The survival rate and body weight were continuously monitored for 14 days.
Coinfection-induced pneumonia experiment: All mice were randomly divided into the following nine groups (6 mice/group): control, H1N1–MRSA coinfection (coinfection), coinfection + HCPM 20 mg/kg, coinfection + HCPM 40 mg/kg, coinfection + HCPM 80 mg/kg, coinfection + HCP 80 mg/kg, coinfection + Oseltamivir 22.75 mg/kg (coinfection + OST), coinfection + Linezolid 200 mg/kg (coinfection + LZD), and coinfection + Oseltamivir (22.75 mg/kg) + Linezolid (200 mg/kg) (coinfection + OST + LZD). All drugs were prepared with 0.5% sodium carboxymethyl cellulose solution. The mice were slightly anesthetized using isoflurane and intranasally instilled with 0.2 LD50 H1N1 solution. The drugs were orally administered to the relevant infected mice 2 h post-infection once daily starting from Day 0 and continuing to Day 4. The uninfected mice were administered 0.5% sodium carboxymethylcellulose solution as the negative control. The mice were next exposed to 107 CFU MRSA intranasally on Day 3. The lungs and small intestines were harvested from the sacrificed mice at 5 dpi for further analysis.
2.8.　 Determination of cytokine production by ELISA
The lung tissues stored at −80 ℃ were thawed on ice, weighed, and homogenized in a tissue homogenizer with sterile PBS (1:9). The contents of the small intestine were removed with ice PBS before weighing, and subsequent sample treatment was performed as with the lungs. The supernatant was collected after centrifugation at 3000 rpm (#TGL-21M, Bioridge Centrifuge, Shanghai, China) and 4 ℃ for 10 min to remove any tissue fragments. Cytokine production in the lung samples and small intestinal tissues was measured by ELISA according to the manufacturer's instructions.
2.9.　 Assessment of bacterial load
To detect the bacterial load in mouse lungs, we prepared a mannitol high-salt agar solid culture medium (MRSA selective) in the biosafety cabinet. Next, lung tissues were homogenized with sterile PBS at a ratio of 1:9. Based on a previously reported study44, the lung homogenate was fivefold gradient diluted and four concentrations (50, 5−1, 5−2, and 5−3) were established. Then, 5 μL of each concentration was spotted on the solid culture medium and incubated at 37 ℃ for 24 h. The total bacterial load in the lungs was calculated.
2.10.　 Histological analysis
To assess pathological damage, the tissues were fixed in 4% paraformaldehyde solution (#BL539A, Biosharp Life Sciences, China) for 48 h and embedded in paraffin. They were sectioned at 5 μm and stained with hematoxylin and eosin (H&E)48. The images were captured under an Olympus VS200 slide scanner (Tokyo, Japan) and analyzed using ImageJ software (ImageJ/Fiji 2.1, National Institution of Health, Bethesda, MD, USA). To assess the expression of complement C3a and C5a in the tissues, immunohistochemical staining was performed as described previously49. The paraffin tissue sections were incubated successively in citrate antigen retrieval solution (#P0081, Beyotime, Shanghai, China) and endogenous peroxidase blocking buffer (#P0100A, Beyotime, Shanghai, China). The sections were then incubated with anti-C3a antibody (1:100) or anti-C5a antibody (1:100) at 4 ℃ overnight, followed by HRP-conjugated secondary antibody (1:50) for 60 min. Detection was done using a DAB horseradish peroxidase color development kit (#P0203, Beyotime, Shanghai, China). Image acquisition was done with an inverted phase contrast fluorescence microscope (Olympus, Tokyo, Japan). Positive staining for complement C3a and C5a was analyzed using ImageJ software.
2.11.　 Small intestinal mucin staining
We used a commercial Alcian blue & nuclear fast red staining kit (#C0153S, Beyotime, Shanghai, China) for mucin staining. Small intestine paraffin sections were deparaffinized in xylene for 10 min, followed by rinsing in fresh xylene for an additional 10 min. The sections were then treated with ethanol in descending concentrations (anhydrous, 90%, 80%, and 70%). After staining with Alcian blue and Nuclear Fast Red solutions, sections were rinsed with tap water for 5 min. Slides were dehydrated in graded ethanol solutions and cleared with xylene before mounting with neutral resin. Imaging was done using an inverted phase contrast fluorescence microscope (Olympus, Tokyo, Japan), revealing blue-stained mucin and pink-stained cell nuclei.
2.12.　 Western blotting analysis
Protein detection was performed by Western blotting as described previously50. Specifically, lung specimens were lysed using RIPA buffer with 1% PMSF to extract total protein. The protein concentration was determined using a BCA assay kit (#P0010, Beyotime, Shanghai, China). The samples were mixed with 5 × SDS-PAGE loading buffer (#P0286, Beyotime, Shanghai, China), denatured at 95 ℃ for 10 min, and separated on 10% SDS-PAGE gels. Following electrophoresis, the proteins (20 μg) were transferred to PVDF membranes. The membranes were blocked and then incubated overnight at 4 ℃ with primary antibodies against Na,K-ATPase α1 (1:1000), H1N1 NP (1:1000), NLRP3 (1:1000), cleaved Caspase-1 (1:1000), Caspase-1 (1:1000), ASC (1:1000), and GAPDH (1:1000). Following incubation with HRP-labeled goat anti-rabbit IgG (H + L) (1:1000) secondary antibody, the signal was detected using the BeyoECL plus detection kit (#P0018S, Beyotime, Shanghai, China) and imaged directly on FluorChem M (ProteinSimple, Silicon Valley, USA).
2.13.　 Quantitative real-time PCR
qRT-PCR analysis was conducted as previously described51. Briefly, RNA was extracted using Trizol reagent (#P2141231, Adamas Life, Shanghai, China), followed by cDNA synthesis using a First Strand cDNA Synthesis Kit (#D7182M, Beyotime, Shanghai, China). Real-time qPCR was performed using 2 × SYBR Green qPCR Mix (#D7265, Beyotime, Shanghai, China) on a real-time PCR instrument (Applied Biosystems, Thermo Scientific, Waltham, MA, USA). The influenza A virus M1 (H1N1 M1) gene were measured using the following primers: forward primer, 5′-AAGACCAATCCTGTCACCTCTGA-3′; and reverse primer, 5′-CAAAGCGTCTACGCTGCAGTC-3′44. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was measured using the following primers: GAPDH forward primer, 5′-ACAGCCTCAAGATCATCAGCA-3′; and reverse primer, 5′-ATGAGTCCTTCCACGATACCA-3′44.
2.14.　 Fluorescence microscopy experiments
Immunofluorescence assays were performed following the instructions of the Multiimmunofluorescence kit (#RC0086-34, Recordbio, Shanghai, China). Paraffin-embedded slides were subject to xylene and ethanol treatment, followed by washing in tap water and PBS. Antigen retrieval was conducted in a citrate solution at 95 ℃, and the sections were subsequently blocked and incubated with primary antibodies. After incubation with secondary antibodies and fluorescent dyes, the slides were mounted with an antifade medium containing DAPI (#P0131, Beyotime, Shanghai, China). The slides were visualized under a laser scanning confocal microscope (Olympus, Tokyo, Japan). The positive areas were identified using ImageJ software.
2.15.　 Preparation of mouse cells for flow cytometry analysis
The spleens, Peyer's patches (PPs), and mesenteric lymph nodes (MLNs) were removed from the mice as previously described52,53. They were minced and ground with a syringe plug. Tissues and cells were collected and filtered through a sterile 70 μm sieve to remove debris. Red blood cells were lysed using a red blood cell lysis buffer (#420301, BioLegend, San Diego, CA, USA) for 5 min. The obtained cell suspension was washed with sterile PBS and resuspended in RPMI 1640 containing 10% FBS (#C0234, Beyotime, Shanghai, China).
Lung lymphocytes were extracted as previously described26. Briefly, lung tissues were minced and digested in prewarmed RPMI 1640 containing 100 U/mL collagenase I (#17100–017, Gibco, Grand Island, NY, USA) and 5% FBS with shaking at 200 rpm for 60 min at 37 ℃. The lymphocytes were enriched by 40%/70% cell separation solution (#17089102, Cytiva, Shanghai, China) gradient centrifugation at 1260×g for 30 min at 4 ℃. The cells at the interface were washed with sterile PBS and resuspended in RPMI 1640 containing 10% FBS.
Lamina propria lymphocytes (LPLs) were extracted as previously described26. Briefly, LPLs were extracted from dissected small intestine sections lacking PPs and MLNs. The intestinal epithelial cells underwent dissociation in RPMI 1640 solution containing 5 mmol/L EDTA (#C0196, Beyotime, Shanghai, China), 1 mmol/L DTT (#ST041, Beyotime, Shanghai, China), and 5% FBS with agitation at 200 rpm for 20 min at 37 ℃. After two rounds of cell dissociation, the remaining segments were minced and digested in RPMI 1640 supplemented with 1 mg/mL collagenase IV (#C5138, Sigma–Aldrich, St. Louis, MO, USA) and 5% FBS, by shaking at 200 rpm for 60 min at 37 ℃. Subsequently, the LPLs were enriched by gradient centrifugation of 40%/70% cell separation solution at 1260×g for 30 min at 4 ℃. The collected cells from the interface were washed with sterile PBS and suspended in RPMI 1640 containing 10% FBS.
The frequencies of Treg and Th17 cells in spleens, lung lymphocytes, LPLs, PPs, and MLNs were determined as previously described26. Before cell staining, the anti-mouse CD16/32 (#abs9477, Absin, Shanghai, China) antibody was used to block Fc receptors. For Treg cells, surface staining with anti-CD4 antibodies (#11-0041-82, eBioscience, San Diego, CA, USA) was followed by intracellular staining with anti-Foxp3 antibodies (#25-5773-82, eBioscience, San Diego, CA, USA) using the Foxp3/Transcription Factor Staining Buffer kit (#00-5523-00, eBioscience, San Diego, CA, USA). Th17 cells were surface-stained with anti-CD4 antibodies and intracellularly stained with anti-IL-17A antibodies (#25-7177-82, eBioscience, San Diego, CA, USA) using an intracellular fixation and permeabilization kit (#88-8824-00, eBioscience, San Diego, CA, USA). Isotype controls were employed for compensation adjustments. The percentages of Treg and Th17 cells were quantified using the flow cytometer (#CytoFLEX S, Beckman Coulter, Pasadena, CA, USA) with data analysis performed using FlowJo software.
2.16.　 Statistical analysis
Statistical analysis was performed using GraphPad Prism 6.0 software. Group comparisons were conducted using a one-way ANOVA with Dunnett's multiple comparisons test. Data are presented as the mean ± standard deviation (SD), with statistical significance set at P < 0.05 (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).
3.　Results
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3.1.　 HCPM and HCP mitigate pneumonia severity in viral-bacterial coinfection mice
The severe pneumonia model induced by H1N1–MRSA coinfection was established during the early stage of our laboratory44. In this experiment, the H1N1 virus infection dose (0.2 LD50) was kept constant44, and the MRSA infection dose was reduced at 108, 107, and 106 CFU. Under the same H1N1 infection titer, the high-dose MRSA coinfected group (108CFU) exhibited a significantly increased lung index (about 15) and stronger histopathological damage at 5 dpi (Supporting Information Fig. S1A and S1B). The other two MRSA groups (107 and 106 CFU) showed similar lung index levels (approx. 12), with only minor surface damage visible in the lungs. However, the lung index for all coinfection groups increased sharply to approximately 19 at 6 dpi (Fig. S1C), with almost the entire lung showing swelling and severe bleeding (Fig. S1A). At this point, the lung index and lung damage response were no longer affected by the MRSA infection dose (Fig. S1A and S1C). The results suggest that secondary MRSA infection after H1N1 infection has a severe impact on the exacerbation of pulmonary inflammatory edema and lung damage during the late stage of infection.
IFN-γ is a typical proinflammatory cytokine that reflects the degree of pulmonary inflammatory damage. Compared with the control group, the high-dose MRSA (108 CFU) coinfection group exhibited a significant increase in IFN-γ expression (Fig. S1D and S1E), which was higher than the other two groups treated with lower MRSA doses (107 and 106 CFU). The results indicate that by keeping the 0.2 LD50 H1N1 infection dose unchanged and reducing the MRSA infection dose, secondary infection with 107 CFU MRSA results in relatively mild pulmonary inflammation and tissue damage, with small differences within the groups. This indicates that the coinfection model is more stable at this dose. In addition, a previous study44 has shown that 108 CFU mice lose more weight and are prone to death during coinfection, which was difficult for collecting sufficient data in the mechanism study.
Based on the above, 107 CFU MRSA was selected to construct the stable coinfection model to explore the efficacy and mechanism of TCM.
Next, we analyzed the differences between H1N1–MRSA coinfection and 0.2 LD50 H1N1 or 107 CFU MRSA infection. The results indicated that H1N1–MRSA coinfection caused more significant weight loss compared with H1N1 or MRSA infection alone (Fig. S1F). Coinfection caused severe lung damage indicated by the severe disruption of the lung architecture, inflammatory cell infiltration, and alveolar wall thickening (Fig. S1G and S1H). In addition, coinfection markedly increased the virus load (Fig. S1I), MRSA colonization (Fig. S1J), and the pro-inflammatory cytokines TNF-α (Fig. S1K) and IFN-γ (Fig. S1L) content in the lungs. In addition, coinfection exhibited severe small intestine damage characterized by disheveled and shortened villi, destroyed crypts, and inflammatory cells infiltration (Fig. S1M). In addition, coinfection showed elevated IFN-γ levels in the gut (Fig. S1N).
Treg/Th17 cell imbalance is a key mechanism associated with a variety of inflammatory diseases. Our results indicated that Treg/Th17 cell imbalance in the gut–lung axis was much more severe in the H1N1–MRSA coinfection group compared with that in the H1N1 or MRSA group (Supporting Information Fig. S2). Therefore, coinfection of H1N1 (0.2 LD50) and MRSA (107 CFU) at extremely low doses leads to severe lung-gut injury, which to some extent reflects the lung-gut symptoms observed in influenza patients with coinfections.
To establish a stable and feasible coinfection model that is better adapted to ethical requirements, mice were infected with 107 CFU MRSA three days following 0.2 LD50 H1N1 infection. The potential efficacy of Houttuynia cordata polysaccharides was examined in the coinfection model.
The in vitro results indicated that both HCPM and HCP had no in vitro anti-H1N1 (Supporting Information Fig. S3) and anti-MRSA (Supporting Information Fig. S4) activities. The 14-day survival results indicated that coinfection mice died from 7 days post virus infection (Fig. 1A). The survival rates in the HCPM (80 mg/kg) and HCP (80 mg/kg) groups were 36% and 10%, respectively. In terms of weight change (Fig. 1B), the body weight of all infected mice decreased after 4 dpi. The HCPM (80 mg/kg) and HCP (80 mg/kg) treatment groups showed a tendency to gain weight after 12 dpi. Collectively, our results suggest that HCPM and HCP can improve the survival rate and body weight of coinfection mice.
As shown in Fig. 1C, HCPM, HCP, Oseltamivir, or Oseltamivir + Linezolid combination treatment decreased the lung index, whereas Linezolid treatment did not show an obvious effect. Coinfection induced remarkable histopathological lesions in the lungs, which was evident by severe disruption of the normal lung architecture, inflammatory cell infiltration, and alveolar wall thickening, all of which were alleviated in the HCPM group, HCP group, Oseltamivir group, and Oseltamivir + Linezolid combination group (Fig. 1D). Damage to Na,K-ATPase α1 activity expression destroys alveolar fluid clearance54. The results indicated that HCPM and HCP significantly enhance the Na,K-ATPase α1 activity (Fig. 1E). Moreover, coinfection mice exhibited much higher viral replication (Fig. 1F) and bacterial load (Fig. 1G), suggesting that coinfection severely impairs host resistance to viral and bacterial infections. However, HCPM or HCP treatment significantly decreased viral replication and bacterial load. Besides, we found that HCPM and HCP significantly inhibited the massive explosion of proinflammatory cytokines, including TNF-α (Fig. 1H), IL-6 (Fig. 1I), IFN-α (Fig. 1J), IFN-β (Fig. 1K), IFN-γ (Fig. 1L), and MPO (Fig. 1M) in the lungs. Taken together, our results indicate that HCPM and HCP without in vitro antiviral and in vitro antimicrobial effects alleviate coinfection-induced pneumonia.
3.2.　 HCPM and HCP alleviate intestinal injury in viral-bacterial coinfection mice
In coinfection mice, we observed not only severe lung lesions; but also small intestinal injury. H&E staining revealed that the control mice did not exhibit obvious histopathological changes (Fig. 2A). However, coinfection mice developed profound enteritis that most strongly manifested as disheveled and shortened villi, destroyed crypts, and inflammatory cell infiltration (Fig. 2A). The pathological lesions of the intestinal tract were markedly ameliorated by HCPM or HCP (Fig. 2A).
Intestinal goblet cells and their secreted mucous substances isolate the intestinal mucosal epithelium from the intestinal contents, which protects the mucosal epithelium. Our results indicated that HCPM and HCP promoted mucin secretion (blue) by goblet cells (Fig. 2B). Tight junctions (TJs) including ZO-1, Claudin-1, and Occludin between intestinal epithelial cells are important components of the intestinal epithelial barrier and inhibit the penetration of luminal antigens, environmental toxins, and bacteria, thus preventing potential focal enteropathy or systemic disease55. The immunofluorescence results indicated that coinfection decreased the expression of ZO-1 (Fig. 2C and F), Occludin (Fig. 2D and G), and Claudin-1 (Fig. 2E and H) in the small intestine, which were restored by HCPM or HCP treatment. In addition, treatment with HCPM or HCP effectively decreased the concentration of the proinflammatory cytokines IL-6 (Fig. 2I), IFN-γ (Fig. 2J), and MPO (Fig. 2K) in the small intestine. Taken together, the results indicate that HCPM and HCP ameliorate intestinal injury in coinfection mice.
3.3.　 The therapeutic effect of HCPM and HCP on intestinal injury precedes lung injury in viral-bacterial coinfection mice
Based on the above studies, we next focused on the dynamic effects of HCPM and HCP on the lung–gut axis. To achieve this objective, we observed the pathological changes in the lungs and small intestines of coinfection mice at 4 dpi and 5 dpi, respectively. As shown in Fig. 3A and B, coinfection caused a certain degree of lung–gut injury as early as 4 dpi. In addition, the severity of lung and small intestine lesions gradually increased with the progression of the coinfection. HCPM and HCP did not show an obvious impact on coinfection-induced lung injury at 4 dpi, but significantly alleviated lung pathology at 5 dpi (Fig. 3A, C, and D). Of note, HCPM and HCP treatment resulted in a significant alleviation in coinfection-induced small intestine injury at both 4 dpi and 5 dpi (Fig. 3E and F). These results indicate that the therapeutic effect of HCPM and HCP on intestinal injury precedes that of lung injury in coinfection mice, which may be attributed to its direct contact with the small intestine in prototype form. Thus, the results suggest that the small intestine may serve as the onset for HCPM and HCP to exert their effects.
3.4.　 HCPM and HCP inhibit the overactivation of intestinal complement in viral-bacterial coinfection mice
The focus of this study was HCPM. When the coinfection model establishment and drug effect were observed, no positive drug group was designed for further mechanistic studies or dynamic observation. Previous studies showed that HCPM exhibits potent anticomplement activity in vitro and in vivo and significantly attenuates H1N1-induced lung injury35. However, it is unclear whether HCPM and HCP can exert beneficial effects in coinfection mice through anticomplement activity. Excessive activation of the complement system occurs during viral or bacterial infection. C3a and C5a are anaphylatoxins produced during the activation of the complement system and participate in the activation of the NLRP3 inflammasome12,18-21. Compared with the control group, the coinfection group showed a significant increase of C3a and C5a in the small intestine as early as 4 dpi, with further increases observed at 5 dpi (Fig. 4). However, HCPM and HCP prominently inhibited the production of C3a and C5a in the small intestine as early as 4 dpi, and this effect persisted until 5 dpi (Fig. 4). Overall, the results indicate that HCPM and HCP inhibit intestinal complement activation in coinfection mice.
3.5.　 HCPM and HCP restrain the overactivation of intestinal NLRP3 inflammasome in viral-bacterial coinfection mice
We examined the activation of the intestinal NLRP3 inflammasome. To determine whether NLRP3 and cleaved-caspase-1 expression change in the gut of coinfection mice, we conducted an immunoblotting analysis of small intestine samples collected from coinfection mice and individuals without coinfection. A significant increase in NLRP3 (Fig. 5A, B, H and I) and cleaved-caspase-1 (Fig. 5A, C, H, and J) expression was observed in the small intestine obtained from coinfection subjects compared with the control subjects from 4 to 5 dpi.
HCPM and HCP treatment nearly eradicated NLRP3 and cleaved-caspase-1 protein expression (Fig. 5A–C and H–J). Next, we examined the fluorescence intensity of NLRP3 and cleaved-caspase-1. Similarly, the immunofluorescence showed that HCPM and HCP produced an inhibitory trend of NLRP3/cleaved-caspase-1 in the small intestine (Fig. 5F, G, M and N). IL-18 and IL-1β are key products of the NLRP3 inflammasome following activation. The results indicated that both HCPM and HCP significantly reduced the release of IL-18 (Fig. 5D and K) and IL-1β (Fig. 5E and L). This effect lasted from 4 to 5 dpi. Collectively, our results indicate that HCPM and HCP may regulate the intestinal NLRP3 inflammasome with inhibiting complement activation in coinfection mice.
3.6.　 The recovery effect of HCPM on Treg/Th17 cell imbalance may be related to its effect on NLRP3 inflammasome signaling in viral-bacterial coinfection mice
Multiple studies have shown that the NLRP3 inflammasome is involved in the imbalance of Treg/Th17 cells23,29,30, which is a key immune mechanism in various respiratory diseases26-28. To further elucidate the role of intestinal NLRP3 inflammasome during coinfection, the NLRP3 inhibitor MCC950 was used to examine the Treg/Th17-mediated immune responses and pathological changes in the gut and lung. The inhibition of MCC950 on intestinal NLRP3 was confirmed by immunoblotting analysis (Supporting Information Fig. S5). Flow cytometry revealed that the coinfection group had a higher frequency of Th17 (CD4+IL-17A+) cells in LPs compared with that in the control group from 4 to 5 dpi, which was decreased by MCC950 treatment (Fig. 6A and B). The NLRP3 inhibitor restored the loss of Treg (CD4+Foxp3+) cells in CD4+ T cells isolated from LPs (Fig. 6C and D). Further analysis revealed that the inhibition of intestinal NLRP3 contributed to the Treg/Th17 cells immune balance (Fig. 6E). In addition, MCC950 also reduced the frequency of Th17 cells in the lungs (Fig. 6F and G), increased the frequency of Treg cells (Fig. 6H and I), and promoting the balance of Treg/Th17 cells (Fig. 6J) in the lungs. More importantly, HCPM also promoted intestinal Treg/Th17 cell homeostasis and further improved pulmonary Treg/Th17 cell homeostasis. Moreover, treatment with MCC950 or HCPM ameliorated the intestinal (Supporting Information Fig. S6A) and pulmonary lesions (Fig. S6B). Thus, HCPM and MCC950 showed a similar effect on coinfection mice. Taken together, these results suggest that HCPM may improve Treg/Th17 cell imbalance in the gut–lung axis along with reducing the NLRP3 inflammasome activation, which contributes to improving the intestinal and pulmonary damage of coinfection mice.
3.7.　 HCPM and HCP regulate the balance of Treg and Th17 cells in the lung and intestinal mucosa during viral-bacterial coinfection
T cells in intestinal mucosa play an important role in regulating the homeostasis of the intestinal environment and affecting other mucosal sites56. Imbalanced Treg/Th17 cells in intestinal mucosa have been reported to be involved in the dysregulation of Treg/Th17 cells in the lungs in a model of viral pneumonia26. Therefore, we subsequently analyzed the changes of Th17/Treg cells in the lungs and small intestines. IL-17A and IL-10 were mainly produced by Th17 and Treg cells, respectively. Compared with the control group, coinfection resulted in a significant increase in IL-17A levels and a marked decrease in IL-10 levels in the lungs and small intestines, which were reversed following HCPM or HCP treatment (Supporting Information Fig. S7). However, IL-17A and IL-10 levels in the small intestines in the Oseltamivir, Linezolid, and Oseltamivir + Linezolid combination groups exhibited a trend of reversion, but there was no statistical significance. Further studies showed that the proportion of Treg/Th17 cells in LPs and lungs was significantly decreased in the coinfection group, suggesting an imbalance of Treg/Th17 cells in the local mucosa of the intestines and lungs, which is a key factor in coinfection-induced gut–lung injury. The administration of HCPM or HCP partially restored the Treg/Th17 cell balance in the LPs (Fig. 7C, D, and F) and lungs (Fig. 7A, B, and E). Oseltamivir or Oseltamivir + Linezolid combination partially reversed the imbalance of Treg/Th17 cells in the lungs, but not in the LPs. Moreover, Linezolid administration had no obvious effect on the imbalance of Treg/Th17 cells in the LPs and lungs. The spleen is the largest peripheral lymphatic organ in mammals and represents global immunity. Coinfection effectively decreased the proportion of Treg/Th17 cells in the spleen (Supporting Information Fig. S8), indicating a disruption of the immune balance in the spleen. However, HCPM or HCP did not affect the Treg/Th17 imbalance in the spleen (Fig. S8). Taken together, these results suggest that HCPM and HCP alleviate gut-lung injury in coinfection mice with modulating the ratio of Treg/Th17 cells in the local mucosa of the gut and lung.
3.8.　 HCPM and HCP restore the frequency of Treg and Th17 cells in the PP–MLN–lung axis in viral-bacterial coinfection mice
The mechanism of action of HCPM was further examined dynamically by measuring the percentages of Th17 and Treg cells in the lung and small intestine at different time points (Fig. 8A–F). After coinfection, the frequency of Th17 cells markedly increased in GALT (PPs and MLNs) and the lungs from 4 dpi. The frequency of Th17 in the PPs and MLNs, and the lungs remained at a high level on 5 dpi. HCPM or HCP treatment markedly reduced the frequency of Th17 cells in the PPs and MLNs from 4 to 5 dpi, and in the lungs on 5 dpi (Fig. 8A, C, and E). In contrast to Th17 cells, the percentage of Treg cells in the PPs, MLNs, and lungs was markedly decreased following coinfection (Fig. 8B, D, and F). HCPM or HCP treatment significantly upregulated the number of Treg cells in the PPs and MLNs from 4 to 5 dpi, and in the lungs on 5 dpi. Taken together, the results indicate that HCPM or HCP treatment restores the balance of Treg/Th17 cells first in the GALT followed by the lung (Supporting Information Fig. S9), which mitigates pneumonia resulting from viral-bacterial coinfection.
4.　Discussion
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Despite the clinical use of antiviral and antibacterial agents for patients with bacterial coinfections, the clinical efficacy of these drugs is not ideal because of the presence of drug resistance and mutated virus46. The advantages of TCM in preventing the progression of mild and moderate COVID-19 cases into severe and critical cases are notable and suitable for immediate application in epidemic prevention and treatment57. Of note, among the components of natural herbs, polysaccharides have emerged as important active constituents due to their prominent immunomodulation activity58. Natural polysaccharides, as macromolecular substances, are not easily absorbed into the blood through the gastrointestinal tract because of a lack of glycosidase for digestion in the human body. Most remain in their original form in the small intestine59-61. Macromolecular polysaccharides, such as HCP and HCPM, which are hard to be absorbed into the blood, have significant anti-complement activities and can attenuate complement-associated diseases11,26,35,62. However, it remains unclear whether HCPM and HCP have anticomplement activities and the potential immunomodulatory mechanisms in virus-bacterial coinfection mice. In this study, we established a coinfection murine model using virus (0.2 LD50 H1N1) and bacteria (107 CFU MRSA) (Fig. S1), and evaluated the therapeutic effects of HCPM and HCP on coinfection mice.
HCPM showed no in vitro antiviral (Fig. S3) and antimicrobial activity (Fig. S4). Our results indicated that HCPM alleviates lung injury in coinfection mice along with the inhibited proliferation of pulmonary viruses and bacteria, the low outbreak of proinflammatory factors, and reduced pulmonary edema (Fig. 1).
Lung injury impairs gas exchange, leading to systemic hypoxia, which in turn causes intestinal ischemia and hypoxia34. This further affects the integrity of the intestinal barrier34. Tight junction proteins (including ZO-1, Claudin-1, and Occludin) and mucin secreted by goblet cells are crucial for the integrity of the intestinal barrier, which is impaired by the changes in these factors63. A similar phenomenon of intestinal barrier impairment was observed in the H1N1–MRSA coinfection mice. However, HCPM or HCP treatment significantly reversed these damages and restored the intestinal barrier (Fig. 2A–H). Furthermore, HCPM or HCP significantly reduced the levels of intestinal inflammatory factors (IL-6, IFN-γ, and MPO) and mitigated intestinal mucosal damage (Fig. 2I–K).
Polysaccharides reach the small intestine primarily in their prototype form63. HCPM and HCP without in vitro antiviral and antibacterial effects could ameliorate coinfection-induced pneumonia, suggesting that their activity occurs through a unique gut–lung pathway. Moreover, we observed that HCPM and HCP exhibited an earlier improvement in the small intestine compared with the lungs (Fig. 3), suggesting that the small intestine is most likely a direct target of HCPM and HCP. More experiments will be done in the future to further confirm the mechanism.
Excessive activation of the complement system occurs during viral or bacterial infections35,64,65. Complement fragments, C3a and C5a, are key anaphylatoxins. Clinical studies16 have shown that anti-C5a antibodies or anti-C3 drugs (such as AMY-101) can improve the symptoms of patients with COVID-19, which gives us a new prospect of targeting the complement system in the treatment of major infectious lung diseases. Our study showed that inhibiting the hyperactivation of C3a and C5a may be beneficial to the coinfection mice (Fig. 4), highlighting the broad potential of complement system therapy.
C3a and C5a activate the NLRP3 inflammasome to amplify inflammation and exacerbate tissue damage18-20. Inhibiting the excessive activation of complement and NLRP3 inflammasome is helpful for the treatment of immunologic injury of intestinal tissue. Existing study66 suggests that drugs that act as blockers of NLRP3 inflammasome signaling may represent innovative strategies for the treatment of intestinal inflammation. In this study, we found that HCPM and HCP exerted a significant inhibitory effect on the excessive activation of the NLRP3 inflammasome (Fig. 5) located in the small intestine. This indicates that the inhibitory effects of HCPM or HCP on NLRP3 inflammasome might be associated with reduced gut complement activation. However, we just observed this phenomenon, and further investigation will be done to confirm the relationship between complement (C3a and C5a) and NLRP3 inflammasome.
The latest study67 has shown that the gut complement system is regulated by the gut microbiota. Our previous study68 showed that the beneficial effects of HCP on viral pneumonia were related to gut microbiota, suggesting that HCPM and HCP might also have a regulatory effect on intestinal flora in coinfection mice. It would be worthwhile to explore the biological links between polysaccharides and gut microbiota, complement and NLRP3 inflammasome in coinfection model.
A recent study69 has revealed a novel communication pathway between the gut and lung, confirming that members of the gut microbiome tune pulmonary immunity through the gut-operated lung immune network (involving ILC2, T and B cells) to promote beneficial disease outcomes in response to pulmonary infections. The gut-lung axis is important for the immune response in respiratory diseases36. Interestingly, immune injury caused by coinfection not only occurs in the lung but also in the small intestine, which further supports the existence of the common mucosal immune system (CMIS). The CMIS protects against invasion of external pathogens in the respiratory and gastrointestinal tract and maintains host immune homeostasis26. T cells in the intestinal mucosa have an important role in regulating the homeostasis of the intestinal environment and affecting other mucosal sites56. Immune cells in GALT (PPs, MLNs), including T cells, migrate from the intestine to the lungs through the CMIS70. The spleen, which is the largest immune organ in the body, contains a substantial number of lymphocytes and macrophages, and serves as the central hub for both cellular and humoral immunity.
As crucial subsets of T cells, Treg cells are significantly reduced in viral- or bacterial-induced pulmonary diseases26,27, whereas excessive activation of Th17 cells exacerbates lung inflammation27,71. Furthermore, an imbalance between Treg and Th17 cells has been observed in the majority of pulmonary inflammatory diseases26-28. More importantly, the imbalance in Treg/Th17 cells in GALT is involved in the dysregulation of Treg/Th17 cells in the lungs26. Related studies have revealed that an imbalance of Treg/Th17 cells is an indicator of COVID-19 severity28. Thus, modulating the balance between these cells, such as through mesenchymal stem cell therapy, may represent a potential treatment for COVID-1972.
Of note, multiple studies have shown that the NLRP3 inflammasome is involved in the imbalance of Treg/Th17 cells23,29,30, which is an important immune mechanism in various inflammatory diseases26-28. This potential mechanism has been rarely reported. A recent study73 indicated that the NLRP3 inflammatory influences Th17 cell development and the conversion of Treg cells into Th17 cells through IL-1β, thus disrupting the Th17/Treg balance and exacerbating tissue inflammation. However, the specific mechanisms require further study. In coinfection mice, we found that direct inhibition of the NLRP3 inflammasome activity using MCC950 reversed intestinal Treg/Th17 cell imbalance and attenuated intestinal injury symptoms (Fig. 6A–E, Fig. S6A). Furthermore, we found that MCC950 restored the balance of Treg/Th17 cells in the lung and further alleviated pulmonary damage (Fig. 6F–J, Fig. S6B). HCPM showed a similar effect as MCC950. In this study, the NLRP3 inflammasome might be the potential target through which HCPM rebalanced Treg/Th17 cells to ameliorate coinfection-induced intestinal and pulmonary injury.
Our results indicate that coinfection causes an imbalance of Treg/Th17 cells in the lung and intestinal mucosa (Fig. 7, Fig. S2), and affects the balance of Treg/Th17 cells in the spleen (Fig. S8), which suggests an effect on CMIS and overall immunity. HCPM or HCP exerted therapeutic effects on coinfection mice, which rectified the Treg/Th17 imbalance in the lung and intestinal mucosa, but not in the spleen, which suggests that HCPM and HCP exhibit immunoregulation through the local mucosa. More importantly, the dynamic changes in the Treg/Th17 cells indicate that HCPM and HCP regulate the dysregulated Treg/Th17 cells in the small intestine prior to the lung (Fig. 8, Fig. S9). Our previous study26 has confirmed that Treg/Th17 cells in the gut-associated lymphoid tissue (GALT) affect Treg/Th17 cells in the lung mucosa during the H1N1 virus infection. Furthermore, the therapeutic effect of HCP on H1N1 virus-induced pneumonia was confirmed to be dependent on the Treg and Th17 cells by gene knockout26. However, the possible mechanism by which the balance of Treg/Th17 cells in GALT affects the balance of Treg/Th17 cells in the lungs of H1N1–MRSA coinfection mice needs to be further investigated. Collectively, these results suggest that HCPM and HCP reduce lung injury in the coinfection mouse model by affecting the NLRP3 pathway and Treg/Th17 cell homeostasis in the gut–lung axis.
Currently, the clinical treatment for influenza virus–bacterial coinfection pneumonia primarily includes a combination of antiviral and antibacterial therapies. Representative antiviral drugs for influenza include Amantadine and Oseltamivir74. Amantadine has been widely used to treat influenza, but its further use has been limited by the rapid emergence of resistance74. Similar concerns have arisen for Oseltamivir75. In addition, the antigenic shift and drift of influenza viruses further increase the challenge of developing effective drugs76. Linezolid or Vancomycin has been used for MRSA infections. However, the emergence of low-susceptibility strains and nephrotoxicity has questioned the status of these therapies as first-line treatments77,78. In addition, the therapeutic window for antibiotic treatment is narrow and difficult to control. In this study, Oseltamivir or Linezolid alone had little effect on the survival rate of coinfection mice. However, Oseltamivir + Linezolid markedly improved the survival rate of coinfected mice (approximately 82%), which was related to their direct targeting effect on the pathogens. TCM has the characteristics of mild effect, multiple targets, low drug resistance, and low toxicity, especially prominent immunoregulation79. HCPM and HCP are natural components derived from Houttuynia cordata in TCM. As shown in Figs. S3 and S4, HCPM and HCP had no direct clearance effect on viruses or bacteria. However, they improved the survival rate of mice (36% and 10%, respectively) as well as coinfection pneumonia to a certain extent. It also suggests that HCPM and HCP play a role in the treatment of infectious lung diseases by regulating the host immune function without being restricted to the type of pathogen. Moreover, we speculated that the difference in efficacy between HCP and HCPM may be caused by the difference in composition, which needs to be further confirmed by subsequent experiments. Polysaccharides are essentially nontoxic and their safety profiles are significantly better compared with that of the combined administration of Oseltamivir and Linezolid80. We found that HCPM and HCP control the severity of coinfection through the regulation of some key inflammation-related targets (complement, NLRP3 inflammasome, Treg/Th17 cells), which provides insight for future drug development targeting related mechanisms. In the absence of targeted drugs for treating drug-resistant bacteria and/or mutated virus-infected lung diseases in the future, plant components may provide some help. Houttuynia cordata has been traditionally used to treat various respiratory tract infections in China. Our study confirmed that HCPM is a key component of Houttuynia cordata for the treatment of drug-resistant bacteria and virus coinfection.
Overall, our results indicate that polysaccharides from Houttuynia cordata exert therapeutic effects on pulmonary inflammatory diseases involving complex pathogen infection through multitarget effects. Nonetheless, there were some limitations to our study. The gut microbiota plays an important role in the intestinal immune response and respiratory system diseases68; however, several questions remain. How does the coinfection change the gut microbiota? Could HCPM or HCP interact with the gut microbiota to help ameliorate pneumonia caused by coinfection? These issues were not specifically addressed in the present study and warrant further investigation.
5.　Conclusions
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Unlike the antibiotics and antiviral drugs that directly against pathogens, anti-complement polysaccharides from Houttuynia cordata exert their therapeutic effects on coinfection mice by modulating the host immune response. Specifically, anti-complement polysaccharides from Houttuynia cordata may regulate the NLRP3 inflammasome and Treg/Th17 cell balance of the gut–lung axis, which contributes to improving coinfection-induced pneumonia. This study has enriched the connotation of the gut–lung axis, and expanded the potential medicinal values of Houttuynia cordata polysaccharides.
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Year 2025 volume 15 Issue 6
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doi: 10.1016/j.apsb.2025.04.008
  • Receive Date：2024-10-11
  • Online Date：2026-04-03
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  • Received：2024-10-11
  • Revised：2025-01-16
  • Accepted：2025-01-27
Affiliations
    aDepartment of Natural Medicine, School of Pharmacy, Fudan University, Shanghai 201203, China
    bDepartment of Biological Medicines, School of Pharmacy, Fudan University, Shanghai 201203, China
    cDepartment of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, China

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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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