마황탕의 인플루엔자 감염 치료 기전 : 마황탕의 지질매개체 발현을 조절해서 면역반응과 염증반응을 컨트롤한다.

[현준]

Effects of maoto (ma-huang-tang) on host lipid mediator and transcriptome signature in influenza virus infection

Nishi A, Kaifuchi N, Shimobori C, Ohbuchi K, Iizuka S, Sugiyama A, Ogura K, Yamamoto M, Kuroki H, Nabeshima S, Yachie A, Matsuoka Y, Kitano H. Effects of maoto (ma-huang-tang) on host lipid mediator and transcriptome signature in influenza virus infection. Sci Rep. 2021 Feb 19;11(1):4232. doi: 10.1038/s41598-021-82707-1. PMID: 33608574; PMCID: PMC7896050.

이 논문은 마황탕이 어떻게 인플루엔자 감염으로부터 바이러스를 이겨낼 수 있게 돕고, 감염반응을 완화시켜서 조직손상을 방지하고 불편감을 줄여서 생존률을 높이는지 실험연구한 논문임.

먼저 쥐 실험을 통해 마황탕 투여가 인플루엔자 감염으로부터 생존률을 높이고 바이러스 수치를 떨어트리며 각종 발현증상들을 완화시킴을 확인하였음.

그리고 쥐의 폐와 혈장을 분석하였는데 그중에서도 Lipid-mediator 지질매개체에 포커스를 맞추었음.

지질매개체는 우리몸에서 세포간의 통신을 전달하거나 면역반응에 활용되는 물질임.

마황탕은 어떤 지질매개체는 양을 늘려주고, 어떤 지질매개체는 양을 줄여주었음. 이러한 양의 증감은 관련 유전형질의 발현율을 조절함으로써 나타남. 즉 마황탕의 항바이러스와 항염증, 면역강화 기전중 하나가 지질매개체의 조절을 통해서 이루어지는 것임.

일반적으로 과도한 염증반응이나 T세포가 많이 발현되어서 과도한 면역반응이 생기는 것을 관련 지질매개체 발현을 줄여서 위 반응들을 조절,억제하였고

inflammation resolution , 즉 염증후 회복기를 촉진하는 지질매개체의 발현과 대식세포와 관련된 지질매개체의 발현을 늘려서 염증의 완화, 조직손상의 회복, 대식세포가 바이러스를 잘 잡아먹을 수 있게 도와주는 경향성이 있었음.

즉, 마황탕은 특정 유전형질의 발현을 조절하여 어떤 지질매개체를 늘리고, 어떤 지질매개체는 줄일 수 있다. 이를 통해 과도한 염증반응을 방지하고 염증해소반응을 촉진하고 대식세포나 T세포를 조절하여 면역반응이 적절하게 이루어지게 할 수 있다.

Abstract

---

Maoto, a traditional kampo medicine, has been clinically prescribed for influenza infection and is reported to relieve symptoms and tissue damage. In this study, we eval‎uated the effects of maoto as an herbal multi-compound medicine on host responses in a mouse model of influenza infection. On the fifth day of oral administration to mice intranasally infected with influenza virus [A/PR/8/34 (H1N1)], maoto significantly improved survival rate, decreased viral titer, and ameliorated the infection-induced phenotype as compared with control mice. Analysis of the lung and plasma transcriptome and lipid mediator metabolite profile showed that maoto altered the profile of lipid mediators derived from ω-6 and ω-3 fatty acids to restore a normal state, and significantly up-regulated the expression‎ of macrophage- and T-cell-related genes. Collectively, these results suggest that maoto regulates the host’s inflammatory response by altering the lipid mediator profile and thereby ameliorating the symptoms of influenza.

마황탕은 전통의학에서 쓰는 처방으로 인플루엔자 감염의 증상과 조직손상을 완화시킨다.

마황탕 투여 실험에서 마황탕은 쥐들의 생존률을 높이고 바이러스 수치를 낮추었으며 감염에 의한 증상발현(발열 통증 등)을 개선시켰다.

마황탕은 오메가3, 오메가6 지방산에서 유래하는 지질매개체의 발현률을 조절함으로써 몸의 정상회복을 돕고 대식세포와 T세포와 관련된 유전자의 발현율을 높여주었다.

마황탕은 인플루엔자에 감염된 개체의 지질매개체의 발현율, 생성량을 조절함으로써 염증반응을 조절할 수 있고 따라서 인플루엔자 감염에 의한 다양한 증상들을 완화시키고 개선시킬 수 있다.

Introduction

---

Epidemic infections of influenza cause half of million deaths every year. Elderly and individuals who have underlying diseases with immune system dysfunction are especially vulnerable to influenza and often develop severe symptoms1,2. Although several anti-influenza drugs targeting influenza viral factors have been developed, problems such as the emergence of strains with acquired drug resistance remain unsolved3. Thus, there is a need for more effective medications to treat influenza virus infection. From this perspective, drug interventions targeted at the host immune response are an attractive approach to suppress the influenza virus and mitigate viral damage without drug resistance.

인플루엔자 바이러스를 공격하는 항바이러스 약물들이 있지만, 이러한 약물들에 내성을 가진 바이러스 변이가 생긴다는 문제점이 있다. 따라서 인플루엔자 감염에서 바이러스 자체를 공격하는 약물이 아니라 감염에 대응하는 개체의 면역력에 관여하는 약물이 가지는 장점이 있다. 이것은 감염에 저항할 수 있게 하고, 바이러스에 의한 손상을 약물내성없이 방지할 수 있다.

Cytokines and lipid mediators are heavily involved in the host response to infection, and appropriate control of these factors is considered to be critical for suppressing the severity of the infection4,5. In the case of infectious diseases, the acute proinflammatory response plays a vital role in host defense by attacking the virus and inhibiting its replication. However, such inflammatory responses also damage tissues, and an excessive response is adversely harmful to the host. Thus, modulating anti-inflammatory effects has long been an important area of research.

급성 염증반응은 바이러스에 대항하는 중요한 반응이다. 하지만 이러한 염증반응은 동시에 정상 신체조직에 손상을 주는 등 과도한 염증반응은 몸에 좋지 않다. 그러므로 염증반응을 적절하게 조절하는 것은 매우 중요하다.

Recent studies have shown that specific lipid mediators appropriately regulate the inflammatory response and its resolution. Furthermore, the role of lipid mediators that promote the resolution of inflammation—in particular, specialized pro-resolving mediators (SPMs) such as protectins, maresins, and resolvins—have been the focus of many studies as endogenous modulators of the inflammatory response during infection6–8. The host response induced by influenza infection involves a complex interaction between the virus and several host factors9; thus, it might be appropriate to use a drug with a long tail to control this type of host response10. Furthermore, a traditional herbal medicine that contains multiple compounds might be an excellent option to simultaneously modulate the host factors.

몇몇 지질매개체들은 염증반응과 염증해결반응을 적절하게 조절할 수 있다. 특히 SPMs 로 protectins, maresins, resolvins 같은 지질매개체들은 염증후 해결반응을 촉진하는 능력이 뛰어나서 이 물질들이 감염에 의한 염증반응을 적절하게 조절하는 내인성 조절인자로 보고 있다.

Maoto (MT; or ma-huang-tang in Chinese), a traditional herbal medicine in Japan (Kampo), is prescribed widely to care for symptoms of upper respiratory infections and influenza11,12. Maoto is a mixture of four component herbs: Armeniacae semen (32.3%), Glycyrrhizae radix (9.6%), Cinnamomi cortex (25.8%), and Ephedrae herba (32.3%). The active ingredients for inflammation response in maoto such as amygdalin in Armeniacae semen, glycyrrhizin in Glycyrrhizae radix, which is metabolized to glycyrrhetinic acid and ephedrine in Ephedrae herba has been reported13–15. Furthermore, we revealed the profile of ingredients in maoto after oral treatment, and we estimated that many ingredients and metabolites of maoto containing ingredients such as pseudoephedrine, methylephedrine, prunasin, liquiritigenin and isoliquiritigenin are absorbed and detected in plasma16. Maoto has antipyretic17,18 and anti-malaise effects in children19, and improves flu symptoms with efficacy comparable to that of neuraminidase inhibitors in adults infected with influenza A virus11,20.

Experimentally, MT has been shown to decrease viral titer and exert an antipyretic effect21,22, as well as to ameliorate influenza virus-induced pneumonia23. The herbal ingredients in MT directly inhibit influenza viral replication in vitro, and affect inflammatory responses both in vitro and in an in vivo animal model14,24–33. While these findings suggest that MT directly inhibits influenza virus infection and regulates the host inflammatory response as a multi-ingredient drug, its detailed mechanism of action has not been clarified.

Studies using in vivo rodent models have shown that MT broadly ameliorates the acute inflammatory response and injury, including both acute lung injury induced by cold/warm cycles of stress34 and asthma in ovalbumin-sensitized mice35. Our previous study revealed that MT significantly ameliorates flu-like symptoms induced by polyinosinic-polycytidylic acid (PIC; a Toll-like receptor 3 agonist) and decreases the pro-inflammatory cytokine response16. We also found that MT influences broad lipid mediator responses. In the acute phase at 2 h after treatment, MT affects the broad ω-3 fatty acid (FA)-derived lipid mediator response associated with anti-inflammatory and pro-resolution responses, and also modulates the acute production of prostaglandins and leukotrienes by PIC. Those findings suggested that MT acts via a specific mechanism to ameliorate acute infectious disease via host lipid mediator and inflammatory systems, specifically affecting anti-inflammatory and pro-resolving factors in the resolution phase of infection.

마황탕은 지질매개체 반응에 광범위하게 영향을 끼친다. 오메가3 지방산 유래 지질매개체의 반응을 변화시킬 수 있다. 이러한 지질매개체들은 항염증과 염증해결반응에 관련이 있다.

또한 마황탕이 감기에서 프로스타글란딘과 류코트리엔의 생성을 조절할 수 있다. -> 과다한 염증반응 조절

In this study, we first eval‎uated the ameliorative effect of MT on a mouse model of influenza virus infection. We then analyzed the lipid mediator and transcriptome profiles on the fifth day after infection to identify endogenous factors affected by MT and to elucidate its pharmacological properties towards influenza virus infection.

Results

---

Maoto reduced viral titer and ameliorated phenotypes induced by influenza virus infection

The experimental protocol is shown in Fig. 1a. We infected mice with mouse-adapted influenza virus [A/PR/8/34(H1N1)] and then orally administered MT daily for 5 days starting 1 h after inoculation. The mortality and phenotype induced by the infection were observed for 8 days post inoculation (dpi).

Figure 1

Effect of maoto on a mouse model of influenza virus infection. (a) Experimental protocol. Influenza virus was administered to mice by intranasal inoculation. Maoto (MT; 0.5 or 2 g/10 mL/kg) was orally administered 1 h after inoculation, and continued for 4 days (5 days treatment in total). Mice with no virus inoculation were used as a control against the infection, and infected mice treated distilled water were used as infected control mice. Data from 10 mice each in the influenza virus inoculation (IVI) control group, IVI with maoto 0.5 g/kg treatment [IVI + MT(L)] group, and IVI with maoto 2 g/kg treatment [IVI + MT(H)] group were used to determine the average survival period. Another 10 mice each in the no-inoculation (NI), IVI, IVI + MT(L), and IVI + MT(H) groups were sacrificed at 5 days post inoculation (dpi) to collect tissue and plasma samples for analysis of viral titer and histopathology. In addition, lipid mediator and transcriptome analysis were performed for the NI, IVI and IVI + MT(H) groups. At 5 dpi, the number of surviving mice were 10, 4, 7, and 9 in the NI, IVI, IVI + MT(L), and IVI + MT(H) groups, respectively. (b) Survival period (dpi). (c) Clinical sign score (dpi). (d) Viral titer. (e) Body temperature. (f) Macroscopic findings. (g) Histopathological score for degeneration and necrosis of bronchial mucosal epithelium. (h) Histopathological images of mouse lung tissue with hematoxylin–eosin staining (× 20). (h1) NI; (h2) IVI; (h3) IVI + MT(L); (h4) IVI + MT(H). * P < 0.01, ** P < 0.05 versus IVI by Log-rank test for survival period, by Mann–Whitney U test with Bonferroni’s multiple comparisons for clinical sign score, macroscopic findings, and histopathological score; and by Welch’s t-test with Bonferroni’s multiple comparisons for viral titer and body temperature.

NI = 대조군으로 인플루엔자 감염시키지 않은 정상 쥐

IVI = 인플루엔자 감염시킨 쥐

IVI + MT = 인플루엔자 감염시키고, 마황탕을 경구투여한 쥐

---

The results showed that MT had anti-influenza virus activity. Whereas all mice in the influenza virus inoculation (IVI) group died by 7 dpi (median survival period, 5 days), those in the group with a higher dose of MT [IVI + MT(H)] lived for significantly longer (median survival period, 7 days) (Fig. 1b). The total clinical sign score [i.e., average summed score of four clinical signs (eye, fur, behavior, and other such as hypothermia, emaciation and respiratory failure), where individual signs were scored from 0 (normal) to 4 (death)], was also significantly reduced by MT treatment (Fig. 1c). The difference in survival rate between the IVI and IVI + MT(H) groups was most significant at 5 dpi. Therefore, we analyzed the effect of MT on viral titer and disease-induced phenotype (body temperature, body weight, lung injury, and pathological findings) at 5 dpi.

At 5 dpi, the number of surviving mice in each group was 4/10 (IVI), 7/10 [lower dose of MT, IVI + MT(L)], and 9/10 [IVI + MT(H)]. The survival rate was significantly higher for the IVI + MT(H) group than for the IVI group. The viral titer at 5 dpi was measured for all surviving mice. The IVI + MT(L) and IVI + MT(H) groups had significantly lower viral titers as compared with the IVI group (Fig. 1d). Regarding the disease-induced phenotype, MT administration significantly increased rectal temperature, which was significantly lowered by IVI (Fig. 1e), but there were no differences in body weight between the IVI and IVI + MT groups (Fig. S1). In addition, MT ameliorated infection-induced severe lung injury (Fig. ​(Fig.1f–h).1f–h). The IVI + MT(H) group had significantly lower scores of macroscopic findings based on an eval‎uation of consolidation area from 0 (no consolidation) to 4 (consolidation area > 2/3) (Fig. 1f). Histopathological analysis showed that MT improved the degeneration and necrosis of bronchial mucosal epithelium induced by influenza infection (Fig. 1g,h; the recorded pathological scores are summarized in Table S1). Taking all observations of the phenotypic effects of MT into account, we used the high MT dose of 2 g/kg at 5 dpi as a measurement point for further analysis of host responses.

Maoto modulated the host lipid mediator response 마황탕이 생체의 지질매개체 반응을 조절한다.

Based on the above effects of MT on the influenza viral titer and host phenotype, we analyzed lipid mediators in lung and plasma by LC–MS/MS in order to elucidate the host response in more detail. In total, the analysis covered 158 lipid mediators, including metabolites derived from ω-3 FA; docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)-derived metabolites; and ω-6 FA-derived metabolites such as prostaglandins and leukotrienes (Table S2).

Overall, 77 lipid mediators were detected in lung. Clustering analysis showed that specific clusters were associated with MT (Fig. 2a and Fig. S2a). Specifically, lipid mediators in clusters A, B and D, which were increased (A) or decreased (B and D) by IVI, were normalized by MT administration. Furthermore, lipid mediators in cluster C were specifically increased by MT. The lipid mediators that were significantly affected by MT are shown in Fig. S2b.

마황탕 투여후 지질매개체의 변화를 분석하였을때 정상군, 인플루엔자군, 인플루엔자+마황탕투여군 세 그룹간의 비교에서

마황탕이 지질매개체의 양을 줄여준 그룹도 있고 늘려준 그룹도 있었다.

a 그림은 쥐의 폐에서 발견된 지질매개체 77개에 대한 3그룹간의 비교입니다.

NI = 대조군으로 인플루엔자 감염시키지 않은 정상 쥐

IVI = 인플루엔자 감염시킨 쥐

IVI + MT = 인플루엔자 감염시키고, 마황탕을 경구투여한 쥐

77개의 지질매개체가 다 동일한 경향성을 띄진 않고 몇개의 구별되는 경향성이 있어서 경향성 별로 그룹화 시킬 수 있었음. A, B, C, D

예를 들어 A그룹은 정상의 건강한 쥐에서는 적게 발현되고 인플루엔자에 감염되면 많이 발현되었음. 그리고 마황탕 투여시 많이 발현되던게 조절되어 적게 발현됨. (= A그룹에 속한 지질매개체는 인플루엔자 감염시에 많이 분비되어 염증과 면역반응에 관여하는데 마황탕이 이 A그룹 지질매개체를 적게 발현시켜 과도한 염증반응을 방지합니다.)

b 그림은 오메가6 지방산 유래 지질매개체 , 오메가3 지방산 유래 지질매개체를 나타낸 그림입니다.

오메가6 지방산 = AA = Arachidonic Acid = 아라키돈산

오메가3 지방산 = DHA, EPA

우측하단 표를 보면 폐에서 나온 지질매개체에서 인플루엔자 감염시 감소하는 비율이 높았고, 마황탕 투여시 지질매개체가 전반적으로 생성 증가 되었다. 70~77.5%

혈장에서 검출된 지질매개체 관련 그림

마찬가지로 정상 쥐, 인플루엔자 감염 쥐, 마황탕 투여 감염쥐 3그룹간의 차이가 있고 마황탕이 지질매개체 생성량에 변화를 주는 그룹들이 있음.

Figure 2

Clustering analysis and metabolic pathway mapping of lipid mediators in lung and plasma. (a) Clustering analysis in lung. A–D show specific clusters of lipid mediators that were altered by maoto (MT) relative to IVI. (b) Pathway mapping of lipid mediators in lung. Log2 fold changes (log2FC) in detected lipid mediators for IVI/NI (left column) and IVI + MT/IVI (right column) were mapped on the metabolic pathway. The percentage of increased or decreased lipid mediators for IVI/NI and IVI + MT/IVI is summarized in the table. (c) Clustering analysis in plasma. A–D show the specific cluster of lipid mediators that were altered by MT relative to IVI. (d) Pathway mapping of lipid mediators in plasma. The log2FC in detected lipid mediators for IVI/NI (left column) and IVI + MT/IVI (right column) were mapped on the metabolic pathway. To show the related pathway of specialized pro-resolving mediators (SPMs), undetected SPMs were included on the pathway as hexagons. The percentage of increased or decreased lipid mediators for IVI/NI and IVI + MT/IVI is summarized in the table. For clustering analysis, the data were normalized and standardized by autoscale for metabolites. Euclidean distance was used for the distance measure, and Ward’s method was used for the clustering algorithm. *P < 0.05, **P < 0.01 versus IVI. ##P < 0.01 versus NI. *P < 0.05, **P < 0.01 versus IVI. #P < 0.05, ##P < 0.01 versus NI.

---

Pathway enrichment analysis of the lipid mediators affected by IVI showed that IVI affected the balance of ω-6 and ω-3 FA-derived lipid mediators. In detail, 63.6% of ω-6 FA-derived lipid mediators and 60.0% of arachidonic acid (AA)-derived lipid mediators were reduced, and 60.0% of ω-3 FA-derived lipid mediators were increased by IVI (Fig. 2b and Fig. S2c). In comparison with the normal state, ω-3 FA-derived lipid mediators suggested activated. By contrast, MT tended to increase both ω-6 and ω-3 FA-derived lipid mediators: specifically, 70.9% of ω-6 FA-derived lipid mediators, 77.5% of AA-derived lipid mediators, and 75% of ω-3 FA-derived lipid mediators were increased by MT. Therefore, MT increased both ω-3 and ω-6 FA-derived lipid mediators, and enhanced the broad lipid mediator pathway in infected mice. This effect of MT seemed to be associated with both activation of the inflammatory response against the influenza virus and resolution of lung damage.

Notably, in the lung, MT significantly increased metabolites in the pro-inflammatory pathway of leukotriene B4 (LTB4), including LTB4 and its metabolites 12-keto-LTB4, as well as prostaglandin J2 (PGJ2), which is involved in protecting the influenza virus infection-associated host immune response5,36,37.

오메가 6 지방산 유래물질중

마황탕은 류코트리엔 B4(LTB4) 및 그 하위 대사경로물질의 생성량을 늘렸음. 인플루엔자 바이러스 감염으로 부터 개체의 면역반응을 보호하는 프로스타글란딘 J2 의 생성량도 늘었음

Furthermore, an ω-3 FA lipid mediator, 10(s),17(s)-dihydroxy-docosahexaenoic acid (10,17-DiHDoHE), which is associated with improvement of acute lung injury in IVI38–40, was significantly decreased by IVI and increased by MT. Therefore, MT enhanced host lipid mediators associated with decreasing influenza virus and with recovery of inflammation-induced tissue damage in lung.

오메가 3지방산 유래물질중 10,17-DiHDoHE의 생성량이 늘어났음. 이 물질은 급성 폐 손상을 개선하는데 도움이 되는 지질매개체임. 결과적으로 마황탕은 폐에서 인플루엔자 바이러스 수치를 감소시키고 염증으로 인한 조직의 손상 회복과 관련된 지질매개체의 생성량을 향상시켯음.

Analysis of the lipid mediator profile in plasma detected 39 lipid mediators, which we subjected to cluster analysis to identify characteristics due to IVI and MT (Fig. 2c and Fig. S3a). Lipid mediators in clusters A and C were decreased and increased, respectively, by IVI, and were normalized by MT. The lipid mediators that were significantly normalized by MT are shown in Fig. S3b. Furthermore, lipid mediators in clusters B and D were specifically increased and decreased, respectively, by MT. In pathway enrichment analysis, 90.9% of AA-derived lipid mediators were increased by IVI. In infected mice, MT tended to reduce ω-6 FA-derived lipid mediators, especially AA-derived lipid mediators, and slightly reduced ω-3 FA-derived lipid mediators. In total, 77.3% of ω-6 FA-derived lipid mediators, 90.9% of AA-derived lipid mediators, and 60% of ω-3 FA-derived lipid mediators were reduced by MT (Fig. 2d, and Fig. S3c). Overall, systemic AA-derived lipid mediators tended to be reduced by MT, which suggests that the effect of MT on host lipid mediators in plasma may act to ameliorate the acute systemic inflammatory response.

마황탕이 개체의 혈장내 지질매개체를 줄여주는 것은, 급성염증에 의한 전신적 반응을 완화시키는 효과가 있는 것과 연관되는것으로 생각된다.

Maoto affected specific gene expression‎ profiles associated with macrophage and T cell immune responses

마황탕은 특정 유전자의 발현을 조절할 수 있다. 이는 마황탕이 대식세포나 T세포의 면역반응 조절과 관련될 수 있다. 즉 대식세포 활성화나 T세포의 활성화와 관련된 유전자가 많이 발현되게 할 수 있음.

Our analysis of the host phenotype and endophenotype for lipid mediators clearly showed that MT acts on the inflammatory immune response of the host. We therefore performed transcriptome analysis to assess the specific gene networks associated with IVI and MT. Weighted gene co-expression‎ network analysis (WGCNA) was used to find the gene networks associated with NI, IVI, and IVI + MT (Fig. S4). In total, we identified 45 modules, among which 9, 17, and 12 modules were significantly associated with NI, IVI and IVI + MT, respectively (Fig. S4e).

Next, we conducted cell-type enrichment (CTen) analysis on the WGCNA-identified modules, which revealed that eight modules were significantly associated with specific cell types (Supplementary Fig. S4f). We also found that three modules (colored-coded as blue, turquoise and green) including relatively large gene sets (blue, 2917; turquoise, 9504; green, 1220), which were significantly enriched in the immune cell marker genes defined in CTen, were correlated with MT, and showed a specific pattern of correlation with each trait (Fig. 3a and Figs. S4g-i). The blue module negatively correlated with NI and positively correlated with IVI and IVI + MT; the turquoise module positively correlated with NI and negatively correlated with IVI and IVI + MT; and the green module negatively correlated with NI, positively correlated with IVI + MT, but did not correlate with IVI. Furthermore, we observed gene sets that were significantly increased/decreased by MT in each module (Fig. ​(Fig.33b–d).

Figure 3

Weighted gene co-expression‎ network analysis (WGCNA). (a) Correlation between module and trait. (b–d) Volcano plot for comparing gene expression‎ between IVI + MT and IVI in the blue, green, and turquoise modules. Blue indicates a significant decrease in gene expression‎ in IVI + MT relative to IVI at P < 0.05 and fold change (FC) < 0.67; red indicates a significant increase in gene expression‎ in IVI + MT relative to IVI at P < 0.05 and FC > 1.5. (e) Summary of cell type enrichment analysis (CTen). –log10 Benjamini–Hochberg adjusted P > 2 was considered to be significant. For each module, the left column shows the results using all genes in the module; the right column shows the results using genes significantly increased by MT relative to IVI. (f) Pathway enrichment analysis of differentially expressed genes between IVI and NI in the blue (FDR < 0.05) (f1) and green (major top 7 pathways with FDR < 0.05) (f2) modules. The gene sets significantly increased by IVI relative to NI at criteria of P < 0.05 and log2FC > 3 were used for analysis. (g) Pathway enrichment analysis of differentially expressed genes between IVI + MT and IVI in the green (FDR < 0.05) (g1) and turquoise (FDR < 0.05) (g2) modules. The gene sets significantly increased by MT relative to IVI at criteria of P < 0.05 and FC > 1.5 were used for analysis.

---

To identify specific gene functions within these three modules, we analyzed the cell type specificity induced by infection and MT by using CTen analysis. Based on all genes in each module (Fig. 3e and Fig. S4j), CTen analysis showed that the blue module was mainly enriched in macrophage cell types, the green module was broadly enriched in many kinds of cell type, while the turquoise module was enriched in lung cell types.

블루모듈 그룹에 속한 유전형질은 대식세포의 생성과 관련되어 풍부하게 있음.

그린모듈 그룹에 속한 유전형질은 여러 종류의 세포유형에 광범위하게 풍부함

청록색모듈 그룹에 속한 유전형질은 폐 세포에 풍부하게 있음.

These results showed that IVI induces a broad host immune response, such as migration/activation of macrophages, and disrupts the normal profile of lung cell gene expression‎ at 5 dpi. By contrast, the genes increased by MT in the blue and green modules were especially enriched in macrophage cell types, while those increased by MT in the turquoise module were mainly enriched in T-cell types. These results suggest that MT notably enhances macrophage and T-cell immune responses in influenza infection (Fig. 3e and Fig. S4j).

마황탕이 대식세포와 T세포의 면역반응을 강화할 수 있다.

Using MetaCore, we conducted pathway enrichment analysis to reveal the module functions associated with influenza infection and MT treatment. This analysis showed that the genes significantly increased by IVI relative to NI (blue and green modules) were mainly enriched in the interferon-γ-associated macrophage activation (blue), neutrophil-derived granule protein and cytokine (blue), aryl hydrocarbon receptor signaling (blue), and interferon signaling (green) pathways (Fig. ​(Fig.3f).3f). On the contrary, no enriched function was observed among the genes with increased expression‎ in the turquoise module (Fig. 3f). Collectively, the results of cell type and pathway enrichment analysis indicated that host responses involving interferons and cytokines in macrophages and neutrophils, which are closely associated with the acute host response in influenza virus infection41,42, continued in inoculated mice until 5 dpi.

We then eval‎uated the effect of MT on these modules. The genes that were significantly increased by MT relative to IVI (green module) were mainly enriched in bradykinin and kallistatin maturation, and in inflammatory responses such as interferon signaling (Fig. 3g1). In addition, the genes that were significantly increased by MT in the turquoise module were primarily enriched in the immune response of the T-cell subset and lectin-induced complement pathway (Fig. 3g2). Furthermore, the gene expression‎ of amphiregulin, which is associated with tissue repair by macrophage and innate lymphoid cells43, was significantly increased by MT [log2FC(IVI + MT/IVI) = 0.858, P < 0.01]. These WGCNA-based data were consistent with the findings from the analysis of differentially expressed genes (DEG) between IVI and NI, and between IVI + MT and IVI (Fig. S5). Notably, key factors such as interferons, lipid mediators, and amphiregulin were increased by MT, which indicates that MT might enhance host responses against influenza virus infection and aid recovery from severe lung tissue damage.

Lastly, we assessed the correlations between gene expression‎ and lipid mediator profiles by WGCNA. For this analysis, the intensity of lipid mediators was used as the trait. As a result, we found lipid mediators that were significantly correlated with the blue, green and turquoise modules (Fig. 4a). The blue and green modules showed a similar correlation pattern, whereas the turquoise module showed an opposite pattern. We then assessed the correlation patterns of the lipid mediators within their metabolic pathways (Fig. 4b). On the one hand, lipid mediators derived from AA and DHA tended to be positively correlated with the blue and green modules. Because these two modules were significantly associated with macrophage gene sets, the lipid mediators positively correlated with these modules may be associated with macrophage function. On the other hand, several lipid mediators metabolized by 5-lipoxygenase (5-LOX) or 15-LOX, 13-hydroxy-octadecatrienoic acid (13-HOTrE), 10,17-DiHDoHE, 15-hydroxy-eicosapentaenoic acid (15-HEPE), 9-hydroxy-octadecadienoic acid (9-HODE), 9-oxo-octadecadienoic acid (9-KODE), 13-HODE, and 15-hydroxy-eicosatrienoic acid (15-HETrE) were positively correlated with the turquoise module. Because the turquoise module is enriched in lung functions, metabolites in the 5- and 15-LOX pathway may be potentially associated with lung tissue function. Furthermore, 12-keto-LTB4, 6-trans-LTB4, 8,12-iso-isoprostane F2 α-VI-1,5-lactone (8,12-iso-iPF2a-VI-1,5-lactone), 8-iso-13,14-dihydro-15-keto-PGF2a, and 14,15-dihydroxy-eicosatetraenoic acid (14,15-DiHETE) were significantly increased by MT relative to IVI and positively correlated with the blue and green modules. Thus, these lipid mediators might be associated with macrophage cells induced by MT.

A그룹에 속한 지질매개체와 B 그룹에 속한 지질매개체가 경향이 다르다.

WGCNA 폐 분석에서 blue, green은 대식세포 발현과 관련이 있었음. 즉 A그룹 지질 매개체는 대식세포 기능활성화와 관련되어 있고 turquoise 그룹 B 그룹에 속한 지질 매개체는 폐기능과 T세포 기능활성화와 관련되어있다.

Figure 4

Correlation between gene expression‎ networks and the lipid mediator profile. (a) Correlation between modules detected by WGCNA (blue, green, and turquoise) and lipid mediator profile. Correlations between the gene expression‎ profile and the lipid mediator profile, which was used as trait, were analyzed by WGCNA. Asterisks indicate lipid mediators that were significantly altered by MT relative to IVI. (b) Pathway mapping of lipid mediators in lung. The WGCNA correlation score of the blue (left column), green (middle column), and turquoise (right column) modules of detected lipid mediators were mapped on the metabolic pathway. Lipid mediators that were detected but not included in the WGCNA modules are indicated by small grey-filled squares. *P < 0.05 for IVI + MT versus IVI by Welch’s t-test with Bonferroni’s multiple comparisons.

---

Discussion

---

Herein, we have confirmed that MT has anti-influenza virus activity and reduces viral titer, consistent with previous studies21–23. We have extended previous research, conducting a more comprehensive analysis of the effect of MT on the host response induced by IVI by examining lipid mediator and gene expression‎ profiles at 5 dpi in a mouse model of IVI with MT treatment as summarized in ​Table1.

Table 1

Summary of effect of maoto on host response in influenza virus infection.

Lipid mediators are associated with and ameliorate pathogenesis in IVI4,5. For example, Tam et al.4 previously examined the changes in lipid mediators in bronchoalveolar lavage during IVI, showing that a lethal infection resulted in an increase in ω-3 FA-derived lipid mediators at 5 dpi, which is consistent with the trends observed in our data. We further showed that MT tended to increase these mediators. Because ω-3 FA-derived lipid mediators generally play a role in modulating inflammation, the enhanced response of the ω-3 FA-derived lipid mediators in the infected state may be part of the host defense response to infection. Therefore, these observations may suggest that MT improves the lipid mediator profile, and this endophenotype seems to correlate with a prolonged survival period.

Our analysis of lipid mediator profiles in lung further identified two types of responses of lipid mediators associated with MT treatment. The first type of response, which involved lipid mediators normalized by MT during the infection, was associated with the IVI-induced immune response (cluster A, B and D in Fig. 2a). The second type of response was associated with an MT-induced specific immune response (cluster C in Fig. 2a). Several lipid mediators increased by MT at 5 dpi were located on pathways involved in the immune response to protect against and reduce influenza viral load, such as LTB436,44, which regulates type 1 interferon signaling and interstitial macrophages36, and PGJ2 as a source of 15-deoxy-delta-13,14-PGJ237. Furthermore, 10,17-DiHDoHE (also known as PDX or protectin D1) improves severe influenza virus infection, bleomycin-induced lung dysfunction and lipopolysaccharide-induced acute lung injury5,38,39. Thus, MT seems to enhance pathways associated with both specific proinflammatory factors and SPMs, which contribute to protection against IVI and resolution of inflammation. Lipid mediators are known to switch class as acute inflammation progresses to resolution. In the early stages of acute inflammation, lipid mediators activate neutrophils to promote phagocytosis, but then switch to recruitment of macrophages. PGE2 and PGD2, which tended to be increased by MT, ultimately activate resolution-type lipid mediators such as lipoxins, resolvins, and protectins, leading to the elimination of inflammation8. Although not all resolution-type lipid mediators were identified, our finding that MT increased 10,17-DiHDoHE at 5 dpi suggests that MT enhances the transition from acute inflammation to resolution. Maintaining the augmentation of lipid mediators may accelerate recovery. Our previous study found that MT is a crude drug mixture that alters many lipid mediators upon intake. Such augmentation of the dynamics of the lipid mediator profile may contribute to the relief of symptoms.

마황탕이 결과적으로 염증 해결과 관련된 지질매개체인 lipoxin, resolvin, protectin을 활성화시키고 증가시켜서 결과적으로 염증의 소멸을 유도할 수 있다.

Regarding the lipid mediator profile in plasma, we observed that IVI caused an increase in AA-derived lipid mediators, which was then reduced by MT treatment at 5 dpi. Because these lipid mediators are generally known as inflammatory factors, MT might suppress systemic inflammation by suppressing such lipid mediators. Several lipid mediators were significantly increased by MT, including 12,13-epoxy-octadecenoic acid (12,13-EpOME), 6-keto-PGF1α, 14,15-dihydroxy-eicosatetraenoic acid (14,15-DiHETE), and 8-hydroxy-docosahexaenoic acid (8-HDoHE), while those decreased by MT were 9,10-dihydroxy-octadecenoic acid (9,10-DiHOME) and 18-hydroxyeicosatetraenoic acids (18-HETE). Although the causes of these alterations by MT are unclear, the lipid mediators identified in plasma may be useful as biomarkers of the MT effect.

마황탕이 단순히 지질매개체의 발현을 증가시키기만 하는 것은 아님. 인플루엔자 감염시 아라키돈산 유래 지질매개체중 일부가 증가하는데 이러한 지질매개체는 일반적으로 염증요소로 알려져 있음. 마황탕은 이러한 지질매개체는 발현을 억제해서 결과적으로 전신적인 염증반응을 억제해줄 수 있음

즉 마황탕은 염증을 유발하는 지질매개체는 줄이고, 염증해결반응을 만드는 지질매개체는 늘려주고 있다.

In our previous study using a rat model of PIC-induced acute inflammation, the prostaglandins and leukotrienes induced in plasma were reduced by MT at 2 h after treatment, suggesting that MT alters the host inflammatory response at an early phase of infection.

이전 연구에서 감염초기의 염증반응의 주요 인자인 류코트리엔과 프로스타글란딘이 마황탕 투여후 혈장내 수치가 감소하였음. 이는 마황탕이 감염초기 과도한 염증반응을 억제해줄 수 있다는 것으로 보임

Because the PIC injection model is considered to partially mimic the host response to virus, it is possible that MT might regulate the host response profile in IVI in a similar manner. That is, in the early phase of viral infection, MT may affect the systemic lipid mediator response for prostaglandins and leukotrienes. Subsequently, in the sub-acute or late phase of the infection, it may modulate the broader ω-6 and ω-3 FA-derived lipid mediator response, providing a comprehensive host immune response and intervening in viral replication. In this study, we only examined the effects at 5 dpi; therefore, we can only hypothesize about the dynamics of the switch in lipid mediator class reflecting the intervention of MT in the host response.

In the transcriptome analysis, gene expression‎ associated with type I interferon, neutrophils and macrophages was induced by influenza infection, consistent with a previous study on transcriptome analysis in IVI45. In the early phase of infection, the phagocytotic activity of macrophages is essential to reduce the virus, and subsequently tissue-resident macrophages play a role in tissue repair. However, prolonged activation of macrophages derived from circulating monocytes promotes alveolar injury46. Our data suggest that the proinflammatory response induced by infection and the enhanced inflammatory response associated with acute lung injury were maintained until 5 dpi.

Cell type enrichment analysis showed that MT upregulated specific genes associated with macrophages and T cells, many of which seemed to be involved in migration and activation. This finding corresponds with that of a previous study of MT (ma huang tang) on H1N1 infection, which showed that MT significantly improved infection-induced lung injury, significantly decreased CD4+ and CD8+ T cells, and increased the ratio of CD4+/CD8+ T cells23. Although the immune cell population needs to be confirmed, these results suggest that MT modulates macrophage and T-cell dynamics, ameliorates the pathology induced by IVI, and repairs lung tissue.

마황탕이 T세포의 발현을 조절할 수 있음. 마황탕 투여후 T세포중 CD4+, CD8+ T세포가 줄었고 또 CD4+ : CD8+ 비율이 늘었음. 즉 CD8+가 특히 더 줄어들었음

T세포가 많이 발현되면 면역반응이 과도하게 일어나서 바이러스를 없애는것뿐만 아니라 폐 조직도 손상될 수 있는데 마황탕이 적절하게 T세포 수를 조절해서 그런 것을 방지하여 폐손상을 예방할 수 있다.

-> 마황탕은 대식세포와 T세포의 활성화, 수의 증가 감소를 조절할 수 있다.

In addition to the interferons and cytokines that MT modulates, we found that MT affect the gene networks for bradykinin and kallistatin maturation. In this pathway, tissue kallikrein is a vital molecule associated with regulation of alveolar macrophages and protection against influenza virus infection47. This pathway would have an essential role in the ameliorative effect of MT.

Furthermore, we found a correlation between the IVI-induced and MT-affected gene networks and lipid mediator response, whereby AA-derived lipid mediators and DHA-derived lipid mediators were positively correlated with the immune response induced by the viral infection, and lipid mediators in the 5-LOX or 15-LOX pathway associated with lung tissue-specific genes tended to be decreased by infection and improved by MT. This result suggests that the dynamics of the lipid mediator profile may reflect an improvement in the pathology. In particular, 12-keto-LTB4, which was positively correlated with a macrophage cell type by CTen, was significantly increased by MT (Fig. 4). Parnet et al.36 previously showed the LTB4 is closely associated with type 1 interferon and alveolar macrophage function, which is consistent with our lipid mediator and gene network analysis of MT. This may infer that MT enhances the LTB4 pathway and affects alveolar macrophage function and the associated gene network pathways such as type I interferon, before shifting the lipid mediator class towards pro-resolution, which leads to its ameliorative effects.

On the other hand, 10,17-DiHDoHE was correlated with the T-cell enriched module, which suggests that 10,17-DiHDoHE may be associated with the effects of MT via the T-cell response and tissue repair. The role of SPMs such as 10,17-DiHDoHE, which was increased by MT, has received much attention8. Furthermore, the metabolic pathway from DHA to 17-HDoHE is closely associated with the production of not only 10,17-DiHDoHE but also other SPMs, resolvins, although the latter lipids were not detected in the present study. These results suggest that MT may tilt the balance towards the pro-resolving phase and accelerate the recovery period in influenza infection by regulating the immune system.

In summary, we have shown that MT has the potential to ameliorate influenza virus infection by altering specific host responses. By modulating the host immune response, MT might be an effective treatment for influenza virus infection that would be free from the problems of drug resistance, unlike other antiviral drugs that directly target viral proteins. In our integrated analysis of lipid mediators and gene expression‎, along with cell type analysis, the group of genes associated with lipid mediators suggested that MT influences the activity of specific immune cells. Thus, the overall effect of MT on host lipid mediator response might be a synergistic action between anti-virus activity and amelioration of the systemic damage due to IVI. In particular, our results regarding the lipid mediators and immune cell groups induced upon infection confirm‎ the observations of previous studies.

Zheng et al. previously reported that the combination of a neuraminidase inhibitor (zanamivir), cyclooxygenase-2 (COX-2) inhibitor (celecoxib), and anti-inflammatory drug (mesalazine) led to an improvement in lethality as compared with a single treatment of zanamivir in cases of IVI with delayed initiation of treatment48. While it will be important to clarify the dynamics of the effect of MT on the host response against IVI in future research, MT affects multiple targets and ameliorates the symptoms of influenza as multi-compound herbal medicine. In addition, the study identified lipid mediators in plasma, which may be potential candidates as a biomarker to verify the effect of crude drug administration, once the connection between lipid mediator profiles in lung and plasma is confirmed. Lastly, although our study confirmed the effect of MT administration on the 5th-day post-infection, there are some limitations of this study. While we elucidated dynamic alteration of gene expression‎ associated with immune response, we further need to reveal the details of immune cells affected by maoto. The mechanism of action of maoto that triggers the host response during the initial administration remains to be eval‎uated by examining a time-dependent analysis of host responses against IVI coupled with MT treatment. Given that maoto is a multi-compound herbal medicine, it is vital to elucidate the synergetic effect of active ingredients in maoto to reveal the specific mechanism of action of maoto as a long-tail drug. Finally, we should integrate the response of the complex host, virus and multi-compound response using systems biology and network pharmacology10,49–51. These approaches have been applied to reveal the complex interaction between endogenous factors and multi-compound of traditional medicines for kinds of disease such as influenza, cancer, type 2 diabetes and rheumatoid arthritis52–56. Applying the approach is valuable to elucidate the overall mode of action of maoto and for repositioning of maoto to related disease. Furthermore, understanding how maoto modulates host inflammation response and immune response gives essential information not only for the anti-influenza virus activity but also therapeutic potential against other acute infectious diseases, and excessive inflammatory response such as cytokine storm.

---