Prophylactic and therapeutic mouse models for evaluating immunologic resilience to infection with influenza virus by Immulina^® (Part 1)

Abstract

Background:

Illness resulting from influenza is a global health problem that has significant adverse socioeconomic impact. Although various strategies such as flu vaccination have beneficial effects, the risk of this illness has not been eliminated. The use of botanicals may provide a complementary approach by enhancement of the host antiviral immune response.

Purpose:

Generate preclinical data using rodent models to determine the most effective utility of a Limnospira (formerly Arthrospira)-derived oral supplement (Immulina^®) for enhancing host immunity to improve antiviral resilience.

Study Design:

Two non-lethal mouse models (prophylactic and therapeutic) were used to evaluate the impact of Immulina^® on increasing host resilience against experimental influenza infection.

Methods:

Mice were fed Immulina^® only for the 2 weeks prior to viral infection (prophylactic regime) or starting 3 days post-viral infection (at the onset of symptoms, therapeutic design). Three doses of Immulina^® were evaluated in each model using both female and male mice.

Results:

Significant protective effect of Immulina^® against viral illness was observed in the prophylactic model (improved clinical scores, less body weight loss, decreased lung/body weight ratio, lower lung viral load, and increased lung IFN-γ and IL-6). Substantially less / minimal protective effect was observed in the therapeutic model.

Conclusion:

This study demonstrates that Immulina^® exerts a protective effect against influenza illness when administered using a prophylactic regime and may not be effective if given after the onset of symptoms. The results will help to optimally design future clinical trials.

Introduction

Influenza (flu) virus infection is an endemic, worldwide public health problem that has challenged humankind for centuries. In the United States, the Center for Disease Control and Prevention has estimated that, between 2010 and 2020, the burden from flu disease resulted in up to 41 million annual illnesses with 12,000 – 52,000 annual deaths (CDC, 2023). The modern flu vaccine program has had significant positive impact by reducing infection risk as well as mitigating flu-induced morbidity and mortality. However, these risks have not been eliminated. Prevention and mitigation success rates vary from year to year, and, more recently (possibly impacted by the COVID-19 pandemic), overall vaccine hesitancy has increased.

The ultimate goal of all vaccine programs is to increase host resilience to the effects of pathogenic microbial agents by preventing or mitigating the effects of infection with agents such as the flu virus. Another approach to improving antiviral resilience against flu and other respiratory viruses is the use of agents that can enhance host immunity to optimize both innate and adaptive antiviral immune responses. Such agents could be particularly valuable for people with chronic immune compromised states such as those with metabolic, autoimmune, or malignant diseases as well as individuals with decreased immunity (e.g., the elderly and very young). To have high potential global health value, such agents should be economical, widely available, and biologically stable for use over long periods of time in diverse environmental conditions.

Limnospira genus (formerly Arthrospira) (Nowicka-Krawczyk et al., 2019) is a cyanobacterium that has been used by many societies around the globe as a food for hundreds of years. Immulina^®, a standardized extract of Limnospira, is a commercial product that contains enriched levels of Braun-type lipoproteins – immunostimulatory compounds that activate the toll-like receptor 2-dependent pathway (Nielsen et al., 2010). Evaluation of Immulina^® in small-scale pilot human studies indicates that daily consumption of this product for 1-week results in increased adaptive immune responses (Løbner et al., 2008) and natural killer cell activity (Nielsen et al., 2010). Additional research (Pugh et al., 2015), using an flu A infection rodent model, suggested that oral administration of Immulina^® mitigates the illness caused by experimental flu A infection. Based on these studies, grant funding was acquired in 2020 to establish the University of Mississippi Botanical Dietary Supplements Research Center (UM-BDSRC), part of the NIH Consortium for Advancing Research on Botanical and Other Natural Products Program. This center is composed of multiple projects investigating the immune antiviral properties of Immulina^® and the data obtained will be used to inform the design of future clinical studies. Completed UM-BDSRC projects include research on product formulation and effect on neutrophils in healthy mice (Li et al., 2023), developed of a chemical standardization method (Huh et al., 2022), and validation of a biological standardization method (Haron et al., 2023). In the current work we report results from a project using an innovative approach to define the optimal regime for Immulina^®’s effect on antiviral resilience. A mouse model of experimental flu A infection was employed, and endpoints included physical parameters (body weight, lung/body weight ratio, clinical illness scores), viral load and levels of eight cytokines. Archived lung histology section from this project were analyzed in an ancillary study to evaluate the effect of Immulina^® on flu A-induced viral pneumonia (Wilson et al., 2023).

Although a growing body of literature indicates that administration of Arthrospira/Limmnospira may have potential value in improving resilience against virus infections, including flu (Capelli and Cysewski, 2010; Pugh et al., 2015; Ratha et al., 2021), it is unclear what treatment schedule/regime would result in optimum antiviral resilience. This would be important to understand when recommending the use of such supplements to optimize antiviral resilience during the season of highest infection rates. The practical application would be based upon determining whether the supplement needs to be taken daily beginning before the season starts (prophylaxis) or would it be expected to have therapeutic benefit in infected individuals prior to the appearance of any symptoms (prodromal) and/or for those presenting with clinical symptoms of viral illness (therapeutic). Answering this question could have significant clinical potential for defining the utility of this supplement and possibly provide a standard for future assessment of other products with putative antiviral properties to maximize resilience. In the current manuscript we present data describing effects of Immulina^® on resilience against flu A-induced illness using two nonlethal rodent models - prophylactic treatment (for prevention of infection) and therapeutic treatment (to minimize severity and duration of the symptomatic phase of infection). These phases represent the most common clinical usage – prevention of infection vs. treatment of clinical illness.

Materials and Methods

Immulina^® extract

Immulina^® (lot 2290020) was acquired from ChromaDex, Corporation (Los Angeles, CA, USA). The composition of Immulina^® powder is 100% extract material (no additives and no preservatives) and is manufactured by extraction of dried Arthrospira/Limnospira with 50% aqueous ethanol at 80°C. Final material is obtained by addition of 1 volume of cold 95% ethanol to the liquid extract and the resulting precipitate is collected by centrifugation, washed with 95% ethanol and dried. Immulina^® extract represents a 10–15% yield, calculated by dividing the dry extract weight by the dry Limnospira weight (used for extraction) and multiplying by 100. Evidence suggests that Braun-type lipoproteins within Immulina^® are the molecules responsible for the in vitro activation of human monocytes (Nielsen et al., 2010). In addition, this crude extract contains high molecular weight polysaccharides (Pasco et al., 2007) and a unique fatty acid profile (palmitic, linoleic and gamma linolenic acids as major constituents and palmitoleic, stearic and oleic acids as minor constituents) (Huh et al., 2022).

Taxonomic identification of the raw material was previously performed using morphological examination and sequence analysis and determined to be Limnospira fusiformis (Huh et al., 2022). Immulina^® is standardized using a bioassay employing THP-1 human monocytes that quantitates potency to ensure batch to batch consistency of activity. Activity in the THP-1 monocyte activation assay is measured by increased expression of an NF-kappa B-driven reporter as previously detailed (Pugh et al., 2001). Immulina^® lot 2290020 exhibited 2508 units of activity/200 mg of extract.

For intragastric administration in mice, Immulina^® was prepared fresh each day by mixing the powdered extract material with water using pestle and mortar by adding small amounts (100 µl) of nanopore sterilized water to reach the desired concentration.

Ethics statement

The research has been carried out in accordance with the ethical and scientific guidelines of The University of Mississippi and the animal research work as per the Institutional Animal Care and Use Committee following the approved protocol # 20–009.

Mice and viral inoculum

C57BL/6 male and female mice (body weights 20–22 g) were purchased from Envigo (Indianapolis, IN, USA). All animals were housed in a pathogenic-free environment with a 12-h light/12-h dark cycle. Throughout the experimental period (two weeks prior and 15 days post-infection) all mice were fed laboratory rodent diet AIN-93M (Research Diets Inc., New Brunswick, NJ, USA).

Flu A virus H1N1, strain A/PR/8/34 was obtained from ATCC, Manassas, VA, USA (VR-1469) and propagated in vitro using Madin-Darby canine kidney cells (as recommended by the supplier). The cells were cultured in Eagle’s Minimum Essential Medium (EMEM; # 30–2003 ATCC) supplemented with 1 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and antibiotics (penicillin 100 units/ml; streptomycin 100 µg/ml) along with 10% fetal bovine serum (#50–152-7078, ThermoFisher Scientific, Waltham, MA, USA). The cells suspended in virus growth medium (EMEM supplemented with 1 mM HEPES, 0.125% bovine serum albumin Fraction V and 1 µg/ml TPCK-treated trypsin) were infected with the virus and passaged several times until viral strength reduced cell viability by 50% (viability determined using Water-Soluble Tetrazolium-8 dye). The supernatant was collected, passed through a 0.2 µm filter, filtrate thus obtained were aliquoted and stored at −80°C until used as the inoculum for infecting mice.

Virus adaptation in mice

The mouse-adapted flu A virus was created using the standard procedure of serial passaging though mouse lungs (Xu et al., 2011). A volume of 50 µl of the tissue-culture prepared inoculum was introduced intranasally into normal mice. The infected mice developed symptoms of respiratory distress within 3–5 days, at which time the mice were sacrificed, and their lungs were homogenized (details provided in the sub-section ‘Preparation of lung homogenates’, Materials and Methods section). A volume of 50 µl of filtered lung homogenate was then inoculated intranasally into a new group of 3 normal mice. After 8 such passages the mouse-adapted virus was inoculated intranasally to another 10 new mice. On day 3 post infection, the lungs of all the infected mice were aseptically removed and homogenized in a total volume of 30 ml. The homogenate obtained was centrifuged and the supernatant passed through a 0.2 µm filter followed by its storage at −80°C in 2 ml aliquots.

The number of infectious units in the inoculum was determined by standard plaque assay (Baer and Kehn-Hall, 2014). Before each experiment the inoculum was diluted to the desired concentration of 5 × 10⁴ plaque forming units per 50 µl for each mouse. Our preliminary experiments had determined that this dose was optimal for a non-lethal viral infection model.

Mouse model

In our preliminary experiments, we observed that 100 mg/kg was the optimum dose of Immulina^® that would not cause any weight loss or any untoward effect in the mice. Based on this, three different doses were selected for evaluation (25 mg/kg, 50 mg/kg, and 100 mg/kg of mouse body weight). All doses were administered intragastrically (by gavage) using a volume of 200 µl containing the desired amount of Immulina^®.

Four studies were conducted that included female mice (prophylaxis treatment model), male mice (prophylaxis treatment model), female mice (therapeutic treatment model), and male mice (therapeutic treatment model). For each study, mice were divided into 5 different groups (with n= 25 for groups I – IV and n=20 for group V). All animals in Groups I – IV were infected intranasally with 5 × 10⁴ infectious units of the virus. Group IV was administered vehicle only (water, no Immulina^®) and served as the infected control mice. Group V comprised the normal control mice and were given vehicle only (water) and were not infected with the virus. For the prophylaxis treatment studies, Immulina^® was administered daily for 14 days prior to viral infection and then discontinued (Fig. 1A). For the therapeutic treatment studies, Immulina^® was administered starting 3 days post-viral infection (average time point for appearance of virus illness symptoms) and continued daily until the end of the study (Fig. 1B). In all studies Immulina^® was orally administered by gavage (25 mg/kg for Group I, 50 mg/kg for Group II, and 100 mg/kg for Group III).

Fig. 1.

Antiviral resilience mouse models used to evaluate Immulina^® – prophylaxis (A) and therapeutic (B).

At 5 timepoints post-infection (days 3, 5, 7, 10, and 15) animals from all groups were weighed and given a clinical score based on observing their condition (1 = normal, 2 = sick, and 3 = moribund). A score of “sick” was defined as slow movement, piloerection, matted fur, and sunken eyes and a score of “moribund” was characterized by slow or no movement, restricted to one place, and no response to touch or noise. Also, at each of the timepoints 5 mice from each group were sacrificed, and their lungs were aseptically removed, weighed, and homogenized. Lung homogenates were aliquoted in 2 ml cryovials and stored at −80°C until analysis of cytokines and viral load. Quantitative analysis of 8 target cytokines (IL-12 p40/p70, IFN-γ, GM-CSF, IL-2, IL-6, IL-10, IL-17A, IL-21) in lung homogenate samples were performed using a bead-based multiplex ELISA technique through testing services offered by RayBiotech (Peachtree Corners, GA, USA).

Preparation of lung homogenates

Each lung sample was homogenized in 4 ml of EMEM (#30–2003, ATCC) containing protease inhibitor cocktail (P2714–1BTL, Millipore-Sigma, Burlington, MA, USA). A stock solution of protease inhibitors was prepared by reconstituting the contents of 1 bottle in 5 ml of nanopure water and the working solution by diluting 250 µl of the stock solution per 50 ml of media. Each sample was homogenized using a plastic disposable generator probe at 4,800 xg using OMNI TH_Q homogenizer. Homogenate was then centrifuging at 4,400 xg for 20 min at 4°C using a Sorvall Legend Mach 1.6 R benchtop centrifuge (ThermoFisher Scientific). The collected supernatant was re-recentrifuged (4,400 xg, 20 min, 4°C) and then the final supernatant was passed through a 0.2 µm filter to remove any tissue residues and bacterial contamination.

Viral quantification by quantitative real time PCR

Viral RNA was extracted from the mouse lung homogenate samples using a DNA/RNA extraction kit from Genesig^® Easy (PrimerDesign Ltd., Eastleigh, UK) following manufacturer’s instructions. The quality and the quantity of the RNA was measured using a Nanodrop (ThermoFisher Scientific). The working stock of each RNA sample was adjusted to 16 ng/µl in sterile Milli-Q water and the final concentration of 80 ng of RNA was used for viral quantification in a real-time PCR per reaction.

The real-time PCR was performed in Bio-Rad CFX connect 96-well thermal cycler (Bio-Rad, Hercules, CA, USA) using One-Step qRT-PCR kit from Genesig^® Advanced kit (PrimerDesign Ltd.), according to instructions of the manufacturer. Primers and probes along with the positive control for the standard curve were available in the kit for viral quantification. The assay condition used were as follows: reverse transcription for 10 min at 55°C and then enzyme activation for 2 min at 95°C followed by 45 cycles of denaturation for 10 sec at 95°C and data collection/capturing for 1 min at 60°C.

Statistical analysis

Differences between groups were analyzed using two-way analysis of variance (ANOVA) followed by Newman-Keul’s Multiple comparison test using GraphPad Prism 10.1.0 software. Minimum criterion for statistical significance was set at p < 0.05 for all comparisons. All data points are presented as the mean ± SEM. *p = 0.01–0.05, **p = 0.001–0.009, ***p = 0.0001–0.0009, and ****p < 0.0001.

Results

Examining the impact of Immulina^® on body weight, the ratio of lung weight to body weight, and clinical infection symptoms

In the prophylactic model (Fig. 1A) oral administration of Immulina^® resulted in a protective effect against a range of physical symptoms (body weight loss, increased lung weight, and clinical symptom scores) stemming from the virus-induced illness. However, when administered according to the therapeutic regime (Fig. 1B), significant restoration effects were mainly observed for clinical signs of infection scores and only minimal effects on restoring normal body and lung weights.

Body weight:

viral infection in all control mice groups resulted in a decline in body weight compared to the uninfected controls, generally between days 3 and 10 (Fig. 2A-B, 3A-B). Upon administration of various doses of Immulina^® a significant prevention of weight loss was observed in the prophylactic design for female mice as compared to the infected controls (Fig. 2A) on day 7 (1.94 ± 1.28 g less at 25 mg/kg, p = 0.0047 and 1.85 ± 2.87 g less at 100 mg/kg, p = 0.0178) and on day 10 (2.59 ± 1.01 g less at 50 mg/kg, p = 0.0053). No significant differences at any dosage level occurred in the therapeutic model (Fig. 2B).

Fig. 2.

Protective effect of Immulina^® against loss of body weight (A, B) and increased lung/body weight ratio (C, D) resulting from flu A (H1N1) viral infection. Female mice were tested in the prophylactic model (PF) and therapeutic model (THF). NC is negative control group (not infected, no Immulina^®), IC is infected control group (infected, no Immulina^®), and 100, 50 and 25 groups refer to mice infected and administered with botanical extract (mg Immulina^® / kg body weight). Time (x-axis) is days post-infection (A-D) and y-axis values represent lung weight/body weight x 100 (C, D). *p = 0.01–0.05, **p = 0.001–0.009, and ****p < 0.0001.

Fig. 3.

Protective effect of Immulina^® against loss of body weight (A, B) and increased lung/body weight ratio (C, D) resulting from flu A (H1N1) viral infection. Male mice were tested in the prophylactic model (PM) and therapeutic model (THM). NC is negative control group (not infected, no Immulina^®), IC is infected control group (infected, no Immulina^®), and 100, 50 and 25 groups refer to mice infected and administered with botanical extract (mg Immulina^® / kg body weight). Time (x-axis) is days post-infection (A-D) and y-axis values represent lung weight/body weight x 100 (C, D). *p = 0.01–0.05, **p = 0.001–0.009, ***p = 0.0001–0.0009, and ****p < 0.0001.

In comparison, the male mice (prophylactic model) exhibited a similar but a more significant protective effect against weight loss across all doses on day 3 with differences of 2.93 ± 2.72 g less (p = 0.0002) at 25 mg/kg, 3.51± 2.21 g less (p < 0.0001) at 50 mg/kg and 2.74 ± 1.54 g less (p = 0.0006) at 100 mg/kg. Significant differences were also observed on day 5 at 25 mg/kg (3.40 ± 3.26 g less, p = 0.0013), 50 mg/kg (5.28 ± 2.34 g less, p < 0.0001) and 100 mg/kg (4.19 ± 1.50 g less, p < 0.0001) as well as for day 7 at 25 mg/kg (4.88 ± 3.03 g less, p = 0.0001), 50 mg/kg (5.95 ± 1.74 g less, p < 0.0001) and 100 mg/kg (5.12 ± 1.99 g less, p < 0.0001) (Fig. 3A). But in the therapeutic model (Fig. 3B) only day 5 for the male mice at the lowest dose (25 mg/kg) showed significantly less weight loss as compared to infected controls (by 1.88 ± 0.66 g, p = 0.0258).

Lung/body weight ratio:

post-infection, a substantial increase in the lung-to-body weight ratio was observed starting on days 3–5 and continuing through day 10 in the infected control groups as compared to the uninfected mice (Fig. 2C-D, 3C-D). For the female mice evaluated using the prophylactic schedule, Immulina^® administration resulted in a significant decrease in the ratio as compared to the infected controls on day 7 (−0.79 ± 0.23% at 50 mg/kg, p = 0.0021, Fig. 2C). In the therapeutic female mice model, although the 50 mg/kg dose resulted in significant reduction on day 3 (−1.06 ± 0.13%, p < 0.0001), we observed an increase in the ratio at this dose on day 7 (0.56 ± 0.69%, p = 0.0233). The 100 mg/kg dose was only effective on day 3 (−0.62 ± 0.49% reduction, p = 0.0098) (Fig. 2D).

In male mice, a significant protective effect of Immulina^® against viral-induced increase in lung/body weight ratio in the prophylactic model was observed on day 5 at 100 mg/kg (−0.44 ± 0.17%, p = 0.0073) as well as on day 7 at 25 mg/kg (−1.11 ± 0.41%, p < 0.0001), 50 mg/kg (−1.29 ± 0.17%, p < 0.0001) and 100 mg/kg (−1.30 ± 0.13%, p < 0.0001). Similar decreases in the lung/body weight ratio were observed on day 10 at 25 mg/kg (−0.40 ± 0.21%, p = 0.0185), 50 mg/kg (−0.39 ± 0.08%, p = 0.0215) and 100 mg/kg (−0.40 ± 0.23%, p = 0.0176) (Fig. 3C). By comparison, in the male therapeutic model a significant reduction in the lung/body weight ratio (Fig. 3D) was observed only on day 5 for all doses: at 25 mg/kg (−0.52 ± 0.18%, p = 0.0026), 50 mg/kg (−0.52 ± 0.14%, p = 0.0024) and 100 mg/kg (−0.44 ± 0.21%, p = 0.014).

Clinical signs of infection:

as expected, we observed increases in signs of illness such as reduced mobility, breathing difficulties, and a generally unwell appearance in the infected control mice, regardless of gender in both the prophylactic and therapeutic treatment designs (Fig. S1 and S2).

For female mice evaluated using the prophylactic model, significant decreases due to Immulina^® administration was observed mainly on day 5 (the average of appearance and mobility scores showed −0.59 ± 0.04 with p values for individual comparisons ≤ 0.0227 at 25 mg/kg, −0.57 ± 0.04 with p values for individual comparisons ≤ 0.0483 at 50 mg/kg and −0.85 ± 0.11 with p values for individual comparisons ≤ 0.0009 at 100 mg/kg) and on day 7 post-infection (the average of all 3 clinical scores showed −0.89 ± 0.17 with p values for individual comparisons ≤ 0.0012 at 25 mg/kg, −0.80 ± 0.06 with p values for individual comparisons ≤ 0.0027 at 50 mg/kg and −0.83 ± 0.16 with p values for individual comparisons ≤ 0.0006 at 100 mg/kg). Although restoration of mobility, respiration, and appearance were also observed in the therapeutic treatment regime, significant effects were only observed at the first two time points post-infection. At day 3 Immulina^® reduced the average of the 3 clinical scores by −0.47 ± 0.19 at 25 mg/kg (p values for individual comparisons were ≤ 0.0393), −0.47 ± 0.11 at 50 mg/kg (p values for individual comparisons were ≤ 0.0023) and −0.72 ± 0.10 at 100 mg/kg (p values for individual comparisons were < 0.0001). At day 5 administration of Immulina^® significantly reduced the respiration score at dose 25 mg/kg (−1.25 ± 0.23, p < 0.0001), the average of respiration and mobility scores at 50 mg/kg (−0.92 ± 0.40 with p values for individual comparisons ≤ 0.0039) and the average of all 3 clinical scores at 100 mg/kg (−0.97 ± 0.37 with p values for individual comparisons ≤ 0.0006) (Fig. S1).

Similar to female mice, the males had a significant reduction in clinical signs of infection scores at more times post-infection due to prophylactic treatment with Immulina^® as compared to the therapeutic regime (Fig. S2). For the prophylactic model, appearance, respiration, and mobility scores were significantly improved with all doses from day 3 to day 7 as compared with the infected controls. The average of the 3 scores for days 3, 5 and 7 showed a reduction of −0.74 ± 0.18 at 25 mg/kg (p values for individual comparisons were ≤ 0.0066), −0.83 ± 0.30 at 50 mg/kg (p values for individual comparisons were ≤ 0.0195) and −0.81 ± 0.26 at 100 mg/kg (p values for individual comparisons were ≤ 0.0066). In contrast, for the therapeutic regime, there was a significant improvement in appearance, mobility, and respiration across all doses on day 5 (the reduction for the average of the 3 scores was −0.49 ± 0.13 at 25 mg/kg, −0.55 ± 0.22 at 50 mg/kg and −0.54 ± 0.21 at 100 mg/kg, p values for individual comparisons were ≤ 0.0015). Similarly on day 7 the average reduction of the 3 scores was −0.97 ± 0.31 (at 50 mg/kg) and −0.67 ± 0.28 (at 100 mg/kg), with p values for individual comparisons ≤ 0.0007.

The impact of Immulina^® on viral load in lung tissue homogenates

In the viral load quantification experiment, it became evident that Immulina^® had a significant impact on reducing the viral load in female mice prophylactic model on days 3 (by 97.57 ± 2.63%, p < 0.0001) and 10 (by 98.62 ± 0.48%, p = 0.0003) at the test concentration of 50mg/kg, as compared to the infected controls. However, no significant differences were observed in the therapeutic model at any dose for the female mice when compared to the infected control group (Fig. 4A-B). Similar results were observed in the male mice. For the prophylactic model, Immulina^® (25 mg/kg) significantly decreased viral load on day 3 (by 88.52 ± 21.06%, p = 0.0193), and for the therapeutic model there was a reduction of viral load on day 5 at 25 mg/kg (by 73.23 ± 26.65%, p = 0.0287) but increased by about 17-fold on day 10 at the 100 mg/kg dose (Fig. 4C-D).

Fig. 4.

Effect of Immulina^® on viral load in female (PF, THF) and male (PM, THM) mice tested in the prophylaxis (A, C) and therapeutic (B, D) models for antiviral resilience. Viral load was determined in lung homogenate samples by quantitative real time PCR. IC is infected control group (infected, no Immulina^®), and 100, 50 and 25 groups refer to mice infected and administered with botanical extract (mg Immulina^® / kg body weight). Time (x-axis) is days post-infection (A-D). *p = 0.01–0.05, **p = 0.001–0.009, ***p = 0.0001–0.0009, and ****p < 0.0001.

The influence of Immulina^® on cytokine levels in lung tissue homogenates

Of the eight cytokines analyzed, IL-6 and IFN-γ were the only ones that were significantly elevated in Immulina^®-treated animals (for at least one timepoint post-infection and for at least one dose), in both genders and both models. For female mice, increased IL-6 (PF, 5.77 ± 3.28 pg/ml, p = 0.0031) and IFN-γ (PF, 11.74 ± 6.82 pg/ml, p = 0.0106 and THF, 6.88 ± 5.24 pg/ml, p = 0.0023) levels occurred primarily on day 3 after infection, especially with a dose of 50mg/kg as compared to control animals (Fig. 5A-D). At day 10 post-infection levels of these two cytokines actually decreased in female mice fed Immulina^® using the administration regime of the therapeutic model (−3.47 ± 1.40 pg/ml, p = 0.0164 at 50 mg/kg for IL-6 and −6.18 ± 1.02 pg/ml, p = 0.0068 at 25 mg/kg for IFN-γ).

Fig. 5.

Effect of Immulina^® on IFN-γ (A, B) and IL-6 (C, D) concentrations in female mice tested in the prophylaxis (PF) and therapeutic (THF) models for antiviral resilience. Cytokine levels were determined in homogenates of lung tissue samples. IC is infected control group (infected, no Immulina^®), and 100, 50 and 25 groups refer to mice infected and administered with botanical extract (mg Immulina^® / kg body weight). Time (x-axis) is days post-infection (A-D). *p = 0.01–0.05, **p = 0.001–0.009.

As for male mice fed Immulina^®, a significant elevation in IL-6 and IFN-γ levels were observed primarily on day 7 post-infection (2 days later than the main effects observed in the female mice). In the prophylactic model (Fig. 6A and 6C), levels of these two cytokines were increased on day 3 at 25mg/kg (4.69 ± 2.90 pg/ml, p = 0.007 for IL-6 and 10.31± 5.76 pg/ml, p = 0.0147 for IFN-γ). On day 7 all doses showed a significant increase for both IL-6 (3.89 ± 3.02 pg/ml, p = 0.0299 at 25 mg/kg, 4.28 ± 2.47 pg/ml, p = 0.015 at 50 mg/kg and 5.15 ± 2.78 pg/ml, p = 0.0028 at 100 mg/kg) and IFN-γ (11.61 ± 7.62 pg/ml, p = 0.0053 at 25 mg/kg, 13.85 ± 8.70 pg/ml, p = 0.0008 at 50 mg/kg and 16.02 ± 8.43 pg/ml, p < 0.0001 at 100 mg/kg). Similarly, in the therapeutic model male mice showed increases on day 7 for IL-6 at 25 and 50 mg/kg doses (4.20 ± 1.35 pg/ml, p = 0.0386 and 7.22 ± 4.41 pg/ml, p < 0.0001, respectively), and IFN-γ at 50 mg/kg (7.09 ± 6.28 pg/ml, p = 0.0004) (Fig. 6B and 6D).

Fig. 6.

Effect of Immulina^® on IFN-γ (A, B) and IL-6 (C, D) concentrations in male mice tested in the prophylaxis (PM) and therapeutic (THM) models for antiviral resilience. Cytokine levels were determined in homogenates of lung tissue samples. IC is infected control group (infected, no Immulina^®), and 100, 50 and 25 groups refer to mice infected and administered with botanical extract (mg Immulina^® / kg body weight). Time (x-axis) is days post-infection (A-D). *p = 0.01–0.05, **p = 0.001–0.009, ***p = 0.0001–0.0009, and ****p < 0.0001.

Analysis of the remaining six cytokines indicated that levels of IL-10, and IL-21 were also increased for female mice fed Immulina^® using the prophylactic model. The effect was primarily observed on day 3 post-infection at 50 mg/kg dose (102.54 ± 50.12 pg/ml, p = 0.003 for IL-10 and 77.02 ± 36.49 pg/ml, p = 0.004 for IL-21) (Fig. S3). However, there were no significant increases in any of the six additional cytokines in the therapeutic treatment model for the Immulina^® treated female mice as compared to non-treated infected controls (Fig. S4).

In the case of male mice for the prophylactic protocol, all six cytokines were increased at the two higher doses on day 7 (average rise of 6 cytokines by 33.91 ± 29.29 pg/ml at 50 mg/kg with p values for individual comparisons ≤ 0.0403 and 40.20 ± 28.54 pg/ml at 100 mg/kg with p values for individual comparisons ≤ 0.005) and the 25 mg/kg dose on day 3 (average rise of 6 cytokines by 33.12 ± 26.06 pg/ml with p values for individual comparisons ≤ 0.0173 (Fig. S5). On the other hand, substantial less effects were observed for male mice treated using the therapeutic design, with significant increases observed only on day 7 at the 50mg/kg dose for GM-CSF (by 3.08 ± 2.26 pg/ml, p = 0.0367), IL-2 (by 3.42 ± 2.34 pg/ml, p = 0.0016), IL-10 (by 33.69 ± 27.97 pg/ml, p = 0.0229), and IL-21 (by 21.04 ± 24.86 pg/ml, p = 0.0056) (Fig. S6).

Discussion

Mouse models have been extensively used as a valuable experimental system to investigate the effects of dietary supplements on modulating host immunity to enhance antiviral resilience. For example, a growing body of research has used a mouse model to demonstrate that administration of probiotic supplements prior to experimental flu viral infection results in a protective effect against viral-induced damage (Zelaya et al., 2016). To establish the model used in the current research, we generated a mouse-adapted flu A virus by serial passage (10 rounds, see Materials and Methods section) of the flu A H1N1, strain A/PR/8/34. The final mouse-adapted inoculum was analyzed with targeted sequencing by the Cell and Microbial Authentication Services provided by ATTC (https://www.atcc.org/services/cell-authentication). The results from this service confirmed that our mouse-adapted virus was the same genus and species/stain as the original human flu A virus purchased from ATTC. All mice were fed a chemically defined rodent chow (AIN-93M) two weeks prior to viral infection and continued throughout the study duration. AIN-93M was selected based on our previous research demonstrating that this chow contains substantially lower levels of macrophage-activating substances (such as endotoxin) as compared with normal rodent diets (Pugh et al., 2015). The objective of using a chemically-defined diet was to minimize the ingestion of chow-derived immunostimulatory substances that could potentially interfere with detection of Immulina^®-dependent effects. In compliance with NIH guidelines for funded research, inclusion of both male and female mice was incorporated into the design of our mouse studies since prior research has reported gender differences in response to severity of infection and outcomes to antiviral treatments (Morgan and Klein, 2019).

Comparison of body weight, lung/body weight ratio, and clinical symptoms in non-infected compared to infected control mice provided functional validation of our experimental model. As expected, viral infection in both male and female mice resulted in significantly decreased body weight, increased lung/body weight ratio and higher scores on clinical signs of infection in the prophylaxis and therapeutic models (NC versus IC groups, Fig. 2, 3, S1, S2). The reduction in weight gain in mice following H1N1 infection is a typical clinical symptom of the disease model, which is well-documented in previous studies (Almutairi et al., 2021; Chandler et al., 2016; Lyu et al., 2020). This observation may be explained by the virus damaging the respiratory system and affecting metabolic pathways, leading to decreased oxygen uptake, increased energy expenditure, and reduced appetite (Shahangian et al., 2021). H1N1 viral infection can cause inflammation and fluid accumulation in the lungs, leading to respiratory distress and increased lung weight (Almutairi et al., 2021).

In our previous research (Pugh et al., 2015), protective effects of Immulina^® administration against loss in body weight and reduced clinical signs of infection were observed in female BALB/c mice using a single oral dose of about 550 mg/kg body weight (diet was supplemented with 0.35% of test material and animals consumed 3.0 – 3.5 g rodent chow/day, representing a daily dose of 10–12 mg of Immulina^®). Our current results from the prophylaxis studies (Fig. 2A, 3A, S1A, S1C, S1E, S2A, S2C, S2E) indicate that these protective effects can also be observed at doses of Immulina^® that are 5 to 20 times lower (100, 50 and 25 mg/kg body wt). The middle dose used in the mouse studies (50 Mg Immulina^®/kg body weight) has a human equivalent dose of about 325 mg for an 80 kg adult (calculation based on allometric scaling, (Nair and Jacob, 2016)). This human equivalent dose is clinically relevant since it is comparable to the average recommended dose of Immulina^® consumption as a dietary supplement (200 – 400 mg/day). Although some statistically significant differences were detected in the therapeutic model (at various timepoints post-infection and doses), the magnitude of effect was substantially less for these parameters (Fig. 2B, 3B, S1B, S1D, S1F, S2B, S2D, S2F).

Although various animal models and human studies (Chen et al., 2016; Lokapirnasari et al., 2016; Markova et al., 2023) indicate that Limnospira exhibits antiviral effects, there are no reports on whether viral load is altered. In our current study, we report that Immulina^®, when administered using a prophylactic regime resulted in a statistically significant reduction in viral RNA levels at various timepoints in both genders (Fig. 4A, 4C). For the therapeutic model, the lack of a significant decrease in viral load observed when Immulina^® was administered to female mice (Fig. 4B) and the increase in viral load in male mice (day 10, Fig. 4D) indicates that Immulina^® may not be an effective agent in enhancing antiviral immune resilience if administered after the onset of viral illness symptoms.

We hypothesize that the protective effect of Immulina^® against flu A-induced illness involves the concentrated presence of Braun-type lipoproteins that mediate host antiviral immune responses. Based on this hypothesis we evaluated the level of eight cytokines in the lung homogenate samples. IFN-γ and IL-6 were the only two cytokines that were significantly increased in both genders and in each of the treatment models. Essentially all of the statistically significant differences in cytokine changes were observed during the first week post-infection (on days 3 and 7), suggesting the immune-enhancing effects of Immulina^® might be maximal at the period of high viral load in the host (by day 10 viral was decreased by several orders of magnitude and by day 15 not detectable).

Previous reports indicate that women have a higher morbidity and mortality risk from flu throughout their lifespan (Cervantes et al., 2022; Morgan and Klein, 2019). In general agreement with this observation, the data from our mice studies indicate that female mice were less responsive to the antiviral protective effects of Immulina^®. Statistically less weight loss and greater reduction in clinical scores (at more time points and more doses) was generally observed for the male mice as compared to female. With respect to immune markers, fewer cytokines were significantly elevated in lung homogenates from female mice and those that were had less doses and less timepoints that reached significance, However, reduction in viral load appear to be greater in the prophylaxis regime for females (days 3 and 10 at 50 mg/kg) as compared to males (only day 3 at 25 mg/kg). If such gender differences are confirmed in future human studies, this may indicate a need for higher doses of Immulina^® in females to achieve similar clinical benefits.

Conclusions

In summary, this study describes an experimental rodent model for nonlethal flu A infection that demonstrates the immunomodulatory effects of intragastric Immulina^® administered prophylactically on the risk for severe disease associated with experimental flu A infection. The mitigating effect provides adequate rationale for the development of similar clinical trials designed to address the impact of daily Immulina^® on clinical measures give before, during or after clinical onset of an active flu infection. Establishing the exact dose(s) and administration schedule(s) for prophylaxis, prodrome and therapy awaits further investigation.

Abbreviations

IC

infected control

EMEM

Eagle’s Minimum Essential Medium

flu

influenza

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

NC

negative control

PF

prophylactic model, female mice

PM

prophylactic model, male mice

THF

therapeutic model, female mice

THM

therapeutic model, male mice