A systematic review on the effects of Echinacea supplementation on cytokine levels: Is there a role in COVID-19?

COVID-19 is the respiratory illness caused by the novel coronavirus, SARS-CoV-2. Cytokine storm appears to be a factor in COVID-19 mortality. Echinacea species have been used historically for immune modulation. A previous rapid review suggested that Echinacea supplementation may decrease the levels of pro-inflammatory cytokines involved in cytokine storm. The objective of the present systematic review was to identify all research that has assessed changes in levels of cytokines relevant to cytokine storm in response to administration of Echinacea supplementation. The following databases were searched: Medline (Ovid), AMED (Ovid), CINAHL (EBSCO), EMBASE (Ovid). Title and abstract screening, full text screening, and data extraction were completed in duplicate using a piloted extraction template. Risk of bias assessment was completed. Qualitative analysis was used to assess for trends in cytokine level changes. The search identified 279 unique publications. After full text screening, 105 studies met criteria for inclusion including 13 human studies, 24 animal studies, and 71 in vitro or ex vivo studies. The data suggest that Echinacea supplementation may be associated with a decrease in the pro-inflammatory cytokines IL-6, IL-8, and TNF, as well as an increase in the anti-inflammatory cytokine IL-10. The risk of bias in the included studies was generally high. While there is currently no substantive research on the therapeutic effects of Echinacea in the management of either cytokine storm or COVID-19, the present evidence related to the herb's impact on cytokine levels suggests that further research may be warranted in the form of a clinical trial involving patients with COVID-19.


Introduction
In early January of 2020, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the agent responsible for coronavirus disease 2019 (COVID-19) [1]. As of June 2021, the global spread of this virus has led to a pandemic with approximately 176 million confirmed cases, including over 3.8 million deaths worldwide [2]. While the majority of COVID-19 patients experience mild to moderate flu-like symptoms (including fever, myalgia or fatigue, and dry cough), severe cases may lead to the development of complications such as acute respiratory distress syndrome (ARDS) and multiple-organ failure [3]. Current scientific literature suggests that "cytokine storm"' is the main cause of ARDS and multiple organ failure in COVID-19 patients [4] through a pathologic process involving excessive inflammation and interference with coagulation leading to clot formation, organ tissue damage (notably in the lungs), multiple organ dysfunction syndrome, septic shock and ultimately death [1,5].
Cytokine storm, also known as cytokine release syndrome, is a Abbreviations: ARDS, acute respiratory distress syndrome; CCL, C-C motif ligand chemokine; COVID-19, coronavirus disease 2019; CSF, Colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; SARS, Severe acute respiratory syndrome; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TFN, tumor necrosis factor. phenomenon observed in response to a number of viral infections and is characterized by a rapid release of pro-inflammatory cytokines [6]. A recent literature review proposed a unified characterization of cytokine storm based on three criteria: "elevated cytokine levels, acute systemic inflammatory symptoms and secondary organ dysfunction beyond that which could be attributed to a normal response to a pathogen, if a pathogen is present'' [7]. Cytokines involved in cytokine storm include proinflammatory interleukin (IL)-6, IL-8, IL-1β, IL-12 and tumor necrosis factor (TNF), while other cytokines such as IL-10 inhibit the process through an anti-inflammatory effect [6]. When considering the role of cytokines in COVID-19 specifically, it has been observed that higher levels of IL-6, IL-8 and TNF, at the time of admission, were associated with significantly lower rates of survival after adjusting for demographics and comorbidities as confounding variables [8]. An association between higher IL-6 and IL-8 levels and increasing disease severity was also observed [8]. In another cohort of COVID-19 patients, highly impaired Interferon (IFN) type 1 response was consistent among severe and critically ill patients [9]. Decreased levels of INF-α and IFN-β were associated with ongoing elevation in blood viral load and an over-active response of pro-inflammatory modulators TNF and IL-6 (9).
Herbal medicines, including species of Echinacea, have been used historically to modulate the immune system. The genus Echinacea has nine different species, with Echinacea angustifolia, Echinacea pallida and Echinacea purpurea commonly employed for medicinal purposes, notably as a treatment for various upper respiratory tract infections and inflammatory ailments [18]. Although the active constituents of the Echinacea genus are well known (e.g., polysaccharides, glycoproteins, caffeic acid derivative and alkamides), their exact mechanism of action is not well understood [19][20][21]. Nonetheless, this herbal therapy seems to be well tolerated with few adverse reactions reported [20].
Previous research indicates that the use of Echinacea may decrease the duration and severity of respiratory tract infections [18], making it a potential candidate to mitigate the symptoms of COVID-19. However, given its ability to stimulate the immune system, there are concerns that using this herb to treat COVID-19 could contribute to or exacerbate the potential for cytokine storm. Interestingly, a recent rapid literature review of clinical trials suggests that Echinacea may have the opposite effect, decreasing pro-inflammatory cytokines and increasing anti-inflammatory cytokines, which may provide a therapeutic benefit in the management of COVID-19 (22). As such, the objective of the present systematic review is to identify all research that has assessed changes in levels of cytokines relevant to cytokine storm in response to administration of Echinacea supplementation.

Search strategy and databases
The following search terms were used: (Echinacea OR Echinacea angustifolia OR Echinacea purpurea OR coneflower) AND (Cytokine* OR cytokine storm OR cytokine release syndrome OR chemokine* OR interferon* OR interleukin* OR tumour necrosis factor* OR colonystimulating factor*). The databases searched included Medline (Ovid), AMED (Ovid), CINAHL (EBSCO), EMBASE (Ovid). The search strategy was informed by an earlier rapid review [22] and conducted on July 14, 2020. An update of the search was conducted on April 12, 2021.

Study selection
Inclusion criteria: 1) administered Echinacea, 2) reported changes in levels of cytokine relevant to cytokine storm (at least one of the following: interferon, interleukin, chemokine, tumor necrosis factor, colony-stimulating factor) and 3) experimental or observational study design, including humans or animals, in vitro/ex vivo studies, and case reports. Exclusion criteria: 1) administration of echinacea in combination with other herbal, medical or nutritional supplements, 2) Reviews, systematic reviews, commentaries, and historical articles. Abstract and full text screening was completed independently in duplicate with any disagreement resolved by consensus. experiments  and 69 studies reported outcomes related to in vitro or ex vivo studies [39,. Tables 1-3 present the characteristics and results of the human, animal and in vitro/ex vivo studies respectfully.
The most commonly studied Echinacea species in human, animal and in vitro/ex vivo studies alike was E. purpurea. Approximately 66% of all studies used E. purpurea alone and another 19% used E. purpurea in combination with other species. The second most commonly studied species was E. angustifolia; with approximately 8% of studies using it on its own and 18% using it in combination with other species.
Human studies were conducted primarily in the USA (38%, n = 5), followed by Italy and Germany (23%, n = 3 each), Indonesia (8%, n = 1) and Ukraine (8%, n = 1). Of the 13 human studies, eight (61%) examined the effects of Echinacea on healthy adults. The remaining five studies examined the effects of Echinacea on: healthy male triathletes training for competition [34], healthy adults exposed to rhinovirus [30], teenagers and adults with new inset of the common cold [28], adults in clinical remission of chronic herpes [35], and COPD outpatients [29]. The largest human study was a clinical trial with 713 participants [28] and the smallest were two non-randomized studies without a control group [39,40] with six participants each. The average number of participants in human studies was 112 (SD = 208) and the median was 40. The Echinacea dosage and duration of treatment employed also varied widely, ranging from a one-time injection containing 5 mg of Echinacea polysaccharides [36] to a daily dose of 8000 mg of Echinacea capsules for 28 consecutive days [32]. A total of four studies [31,32,34,37] implemented 28-day interventions and three employed a one-time dose [36,38,40]. Concerningly, two studies [33,35] did not specify the dosage of Echinacea used. Moreover, Echinacea tablets or soft gel capsules were the most common type of intervention. Additional interventions included Echinacea lozenges, syrup, juice and tinctures. All of the human studies except for one [31] assessed changes in interleukins, with IL-6 being the most common, closely followed by IL-8, IL-1B, then IL-10, IL-2, IL-12 and IL-3. The second most commonly studied cytokine was TNF (61%, n = 8). Lastly, three studies (23%) assessed changes in INF and only one (8%) assessed changes in GM-CSF. None of the human studies included assessed changes in chemokines.
Animal studies were conducted in mouse or rat models, although studies also included dogs [54], tilapia [45], and guinea pigs [55]. Sixteen trials had a duration of at least two weeks while five lasted four to seven days [41,43,50,57,131] and three lasted one day or less [60,61,63]. The daily dose of Echinacea varied widely from 5 to 500 mg/kg per day.
The cell culture studies used a variety of immune cells. Immune stimulation was achieved through a variety of methods; the most common where exposure to LPS (n = 29), viruses (n = 14) and phytohemagglutinin and/or phorbol 12-myristate 13-acetate (n = 10). Studies assessed changes in the amount of cytokines produced or changes in genetic expression following exposure to Echinacea.

Change in cytokine levels
The changes in cytokine levels that followed Echinacea supplementation are presented in Fig. 2. Results are presented for the cytokines relevant to the progression of cytokine storm. Among the human studies, decreased levels of the pro-inflammatory cytokine IL-6, IL-8, and TNF were reported by 57, 50, and 62% of studies that measured these cytokines, respectively. Among the animal studies decreased levels of proinflammatory cytokines IL-1, IL-6, and TNF, were reported by 73, 78, 74% of studies that measured these cytokines, respectively. However, increased levels of the pro-inflammatory cytokine IL-2 were reported by 57% of animal studies. In addition, an increase in levels of the antiinflammatory cytokine IL-10 were reported by 57% of animal studies that measured this cytokine. Among the cell culture studies, decreased levels of pro-inflammatory cytokines IL-6, IL-8, CCL2, CCCL3, and CCL4 were reported by 63, 70, 67, 75, 71% of studies that measured these cytokines, respectively. Moreover, nearly two thirds of the cell culture studies that measured levels of the anti-inflammatory cytokine IL-10 reported an increase. IFN levels were increased in the majority of human, animal, and cell culture studies; while this cytokine is considered to be pro-inflammatory, decreased levels of IFN have been detected among COVID-19 patients. None of the studies reported cases of cytokine storm.

Risk of bias assessment
The results of the risk of bias assessments for the human RCT and non-RCT studies are presented in Figs. 3 and 4. In total, six of these studies had a "high risk of bias", two studies had "some concerns" or "moderate risk of bias" and two studies had "low risk of bias". Among the pre-post human studies, two received a rating of "fair" and one received a rating of "poor". Among the animal studies, each one received a rating of "probably high risk of bias" in at least one category. Three received a rating of "definitely high risk of bias" in one category. Additional information on the risk of bias assessment for the pre-post and animal studies is found in Supplemental File 2. Among the cell culture studies, thirty-eight (55%) received as score of 1 corresponding to "reliable without restrictions". Thirty-one (45%) received a score of 3 corresponding to "unreliable".

Discussion
The present systematic review identified all human, animal, and cell culture data reporting the impact of Echinacea supplementation on cytokine levels. The data suggest that Echinacea supplementation may be associated with a decrease in the pro-inflammatory cytokines IL-6, IL-8 and TNF as well as an increase in the anti-inflammatory cytokine IL-10. In addition, it may be associated with an increase in IFN, a proinflammatory cytokine reported to be low in patients with COVID-19. Overall, the findings of the human and animal studies were more likely to report primarily anti-inflammatory effects. Ex vivo and in vitro studies demonstrated more of a mixture of pro-and anti-inflammatory effects; however, given that they were conducted in the isolation of cell culture rather than in the context of a highly complex, functioning immune system, the results may be less relevant to use in humans. The findings suggest that the use of Echinacea supplementation may be useful in the prevention or management of COVID-19-related cytokine storm in humans, however further targeted studies are needed.
Levels of IL-6 and TNF both independently predict COVID-19 disease severity and mortality [8] and may be important therapeutic targets. Therapies aimed at inhibiting these cytokines have demonstrated improvements in the clinical course of severely ill COVID-19 patients. A meta-analysis of studies administering the IL-6 receptor monoclonal antibody tocilizumab to patients with severe COVID-19 revealed a reduction in mortality and the need for mechanical ventilation [132]. The effects of other immunomodulatory agents including anakinra, an inhibitor of IL-1, and sarilumab and siltuximab, inhibitors of IL-6, were inconclusive [133]. Observational registry data from patients with inflammatory bowel disease who contracted COVID-19 suggest a possible benefit from taking anti-TNF medication in terms of a composite outcome of death or hospital admission, however not with either outcome alone [134]. A call to prioritize the study of anti-TNF therapy has been made [134]. Because IL-6 and TNF are independently associated with clinical outcomes, it has been hypothesized that therapy targeted at the inhibition of both cytokines simultaneously may yield additional benefit and warrant study [8]. Echinacea may decrease production of these two cytokines.
Among the studies identified in the present review, more studies reported an increase in IFN production than a decrease following Echinacea supplementation. While IFN-α and β are considered proinflammatory in nature, they also play a critical role in exerting an antiviral effect. Observation of depressed levels of IFN-α and β among COVID-19 patients has occurred [9]. While the trial reporting this finding was primarily cross-sectional, sequential assessment found that the depressed levels of IFN-α preceded worsening of disease severity and transfer to more intensive care [9]. The virus SARS-CoV, the causative agent of severe acute respiratory syndrome (SARS), inhibits production of IFNs in order to diminish the innate immune response of the host [135]. A need to explore therapeutic approaches to increase IFN in the treatment of COVID-19 has been proposed [9].
Additional evidence that may be considered regarding the potential usefulness of Echinacea in the management of COVID-19 include the herb's ability to decrease the severity and duration of acute respiratory tract infections [22] and in vitro data demonstrating direct antiviral effect of Echinacea against several coronaviruses including SARS-CoV-2 ( [136]).
The present review has several strengths and limitations. Strengths of the review include a rigorous search strategy that was conducted in multiple databases, as well as duplicate screening and data extraction. The review process is limited by a high level of heterogeneity among the included studies and subsequently, the inability to complete metaanalysis. The findings are limited by the high risk of bias found in many of the included studies. They are also limited by the fact that none of the studies assessed the impact of Echinacea on cytokine changes in patients or models of COVID-19. Many of the human studies involved healthy participants or participants with relatively mild infections such as the common cold. The animal and cell culture studies used a variety of immune stimulating agents such as lipopolysaccharide (LPS), bacterial and viral infections. While animal models of cytokine storm exist [137], none were used by the studies included in the present review. These factors may decrease the generalizability of the findings to the treatment of COVID-19.
Similarly, the studies did not assess the changes in cytokine levels in models of cytokine storm. Cytokine storm is a complex syndrome involving cascades of interdependent inflammatory mediators which changes over the course of clinical progression. Defining this condition has been challenging due to the difficulty of differentiating a dysregulated immune response from a physiologic response to a severe infection [7]. Cytokines play an important role in the host response to an infection but at the same time, may cause harm to the host when released in excess. It has been hypothesized that inhibition of cytokine signaling could impair clearance of SARS-CoV-2, and result in worse outcomes such as secondary infections; this has been previously observed in the treatment of influenza [138] and subsequent to the use of IL-6 inhibitors in COVID-19 patients [133]. These findings may suggest that immune modulation may be appropriate for only a subgroup of COVID-19 patients. Additionally, cytokine production varies over the course of the response to the pathogen. Ideally, the immune response should be proportionate to the severity of the infection and result in a return to homeostasis following clearance of the pathogen [7]. The importance of timing may be relevant to interpreting the findings of the present review. The included studies measured cytokine levels at a variety of    Only E. angustifolia and E. pallida-treated mice demonstrated statistically significantly higher production of IL-4 (p = 0.046) and increased IL-10 production (p = 0.057) -no effect on IL-6 by any of the preparation -Statistically significantly increased IL-2 (p < 0.035) -no effect on IL-12 production -Statistically significant inhibition of TNF-α production from splenocytes from all 3 preparations. (p = 0.004) Zhang, 2020 [61] National               timepoints in the course of an infection; the impact of timing may account for some of the heterogeneity in the results presented. It has been hypothesized that the cytokine storm seen in COVID-19 occurs in two stages. The first stage is an underactive initial immune response which fails to adequately clear the virus. Subsequently, in response to the failed clearance, there is an overactive immune response [139]. Changes in the immune response at different time points in the course of disease progression suggest that the timing of different immunomodulatory therapies may be highly important [139].

Conclusion
The findings of the present systematic review suggest that the effect of Echinacea supplementation on cytokines may be predominantly antiinflammatory, including the inhibition of cytokines that play a key role in the progression of severe COVID-19. Investigation of the potential therapeutic role of Echinacea supplementation in the prevention or treatment of cytokine storm due to COVID-19 may be warranted.

Funding
No funding was received for the conduct of this research.

Author contributions
The project was conceived by MA, KC and VC. MA, KC and VC developed the study protocol. The search strategy was conducted by VC. Data extraction was completed by all authors. Preliminary data analysis was completed by MA. All authors contributed to manuscript preparation and approved the final manuscript draft.

Declaration of competing interest
The authors declare no conflict of interest.