Generation of Rgs10-deficient mice (Rgs10-/-) was described previously (18). Briefly, mice were backcrossed on a C57BL/6 genetic background for 10 generations. All Rgs10-/- mice and their wild-type (WT) C57BL/6 littermates were maintained in specific, pathogen-free animal facility, bred and housed in individually ventilated cages, with free access to food and water on campus in the Coverdell Vivarium at the University of Georgia. Pups were weaned from the mothers at a standard 21 days of age unless they appeared unable to support themselves, in which case they were weaned at 28 days. Genotyped animals were age- and sex-matched, randomly allocated to experimental groups, and randomly assessed. For identification purposes, standard ear-tagging was done. Animals were euthanized with CO₂ and subsequent cervical dislocation when appropriate. No experimental drugs were used in this study. The following PCR primers were used to genotype mice:

Rgs10 forward: 5’-CCACGAGGAAGTGAAGTGAAAGCTTT-3’,

Rgs10 reverse: 5’-AGTCAGTTCTGAGTGTGTGAAAGTGC-3’, and

LTR2: 5’ AAATGGCGTTACTTAAGCTAGCTTGC-3.

The expected product size for the WT band is 400 bp and that for the Rgs10-/- is 200 bp.

Equal number of female and male WT and Rgs10-/- mice, which were studied at eight- to ten-weeks of age, were anesthetized by intraperitoneal injection of Avertin (2,2,2-tribromoethanol (TBE)) (250 mg/kg). A negative response to a toe pinch confirmed adequate anesthetic depth. Mice were then inoculated intranasally (i.n.) with 100 plaque-forming unit (PFU) of mouse-adapted PR8 (A/Puerto Rico/8/1934 (H1N1)) influenza A virus in 50 µl of 1X-phosphate buffered saline (PBS). The infected mice were monitored daily for weight loss. In accordance with institutional guideline at the time of conducting the experiments, mice that lost >20% of their body weight were considered moribund and thus euthanized. All the animal experiments and the procedures were approved by Institutional Animal Care and Use Committee at The University of Georgia (protocol ID: A2020 11-001-Y1-A3).

The Madin-Darby canine kidney (MDCK)-Atlanta cell line is a generous gift from Dr. Stephen Tompkins lab at University of Georgia (Athens, GA). MDCK-Atlanta cells were grown in HyClone Dulbecco’s Modified Eagle Medium (DMEM)/F12 1:1: Liquid (Cytiva, cat#: SH30126.01) supplemented with 10% BenchMark™ heat-inactivated fetal bovine serum (Gemini Bio, cat#: 100-106), 1% of L-Glutamine Solution (Gemini Bio, cat#: 400-106), and an antibiotics combination of (1% penicillin/streptomycin) (Gemini Bio, cat#: 400-109) and incubated at 37°C in a humidified atmosphere 5% CO₂.

The A/Puerto Rico/8/1934(H1N1) influenza virus strain (referred to as PR8) was grown in the allantoic cavity of 9- to 10-day old specific pathogen-free embryonated chicken eggs at 37°C. The allantoic fluids were collected and then cleared by low-speed centrifugation. The viral supernatants were subsequently overlaid onto a 25% sucrose cushion in NTE buffer (100 mM NaCl, 10 mM Tris, 1 mM EDTA) and pelleted by ultracentrifugation in a Beckman Coulter SW32 rotor at 28,000 rpm for 2 hours. The purified PR8 viruses were resuspended in 2 mL of 1XPBS buffer, aliquoted, and frozen at − 80 °C. The viral stock titer was determined by plaque assay on monolayers of MDCK-Atlanta cells and hemagglutination assay, as described previously (25).

The mice were first anesthetized and euthanized. After the trachea was exposed, the airways of euthanized mice were washed three times with 1 ml of sterile PBS, and the collected BALF was centrifuged at 400 x g for 10 min at 4°C. Soluble protein analytes of mouse BALF from WT and Rgs10 -/- mice were collected and subsequently preserved with addition of 1 mM PMSF and 1X proteases/phosphatase inhibitor cocktail (Cell Signaling technology, cat#: 5872S) before storage at − 80 °C for protein, chemokine, and cytokine detection. The concentration of proteins was measured using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, cat#: 23225).

After lysing red blood cells using ammonium-chloride-potassium (ACK) lysis buffer (Thermo Fisher Scientific, cat#: A1049201) and two 1X PBS wash step, leukocytes in the BALF pellet were resuspended in 1 ml sterile 1X PBS and counted. For phenotyping, 1X10⁶ cells were suspended in 100 µl of 1X PBS and stained with Zombie Aqua™ flexible viability dye (1:100, BioLegend, Cat#: 423101) at room temperature in the dark for 15 mins. Following one washing step, the cells were suspended in 100 µl of 1X PBS containing (1% bovine serum albumin) and blocked using TruStain FcX™ anti-mouse (CD16/32) antibody (BioLegend, Cat#: 101320) on ice and protected from the light for 10 mins. The cells were subsequently stained with the following surface antibodies: ‘myeloid antibody cocktail’ [CD11b-PE/Cy7 (Cat#: 101216), CD115-APC (Cat#: 135510), CD11c-BV605 (Cat#: 117334), Ly6G-AF488 (Cat#: 127626), Ly6C-APC/F750 (Cat#: 128046), and F4/80-PE (Cat#: 123110)] and ‘lymphoid antibody cocktail’ [CD45-APC/F750 (Cat#: 103154), CD3-APC (Cat#: 100236), CD4-AF488 (Cat#: 100423), CD8a-AF700 (Cat#: 100766), B220-BV785 (Cat#: 103246), and NK 1.1-PE (Cat#: 108708)], all from Biolegend (San Diego, CA) at a 1:100 dilution at the same time to detect the number of cells from each of the myeloid and lymphoid cell subsets. Samples were read within 12 hours using a NovoCyte Flow Cytometer (ACEA Biosciences, Inc.) with the NovoExpress^® software at the University of Georgia College of Veterinary Medicine Cytometry Core Facility. Data acquisition was followed by analysis using FlowJo v.10.6.1 (BD Biosciences, San Jose, CA).

The magnetic bead-based Bio-Plex assay was used to measure levels of 31 different mouse cytokines and chemokines in the BALF (stored at -80°C as indicated above) using internal standards (Bio-Plex Pro Mouse Chemokine Panel 31-plex, Bio-Rad, Cat#:12009159) following manufacturer’s instructions. The assay was performed at the University of Georgia CTEGD Shared Resources Laboratory on a Bio-Plex 3D analyzer (Bio-Rad Inc., Hercules, CA). Interferon-alpha enzyme-linked immunosorbent assay kit (Invitrogen, Cat#: BMS6027) was used to measure INFα levels in the BALF following the manufacturer’s protocol.

Lung tissues from infected WT and Rgs10 -/- mice were collected and homogenized in 1 ml 1X PBS using GentleMacs Dissociator (Miltenyi Biotech, Cat#: 130-093-235). Lung homogenates were centrifuged at 5,000 x g for 15 mins at 4°C, and virus-containing supernatants were collected and stored at -80°C. Lung viral titers were determined by plaque assay on MDCK-Atlanta cells that were infected by serial, 10-fold dilutions of infected lung homogenate supernatants in DMEM/F12 (1:1) medium-containing 1 µg of trypsin, TPCK treated (Worthington Biochemical, Cat#: LS003740). Following 1 hour of MDCK-Atlanta cells infection, prepared overlay medium containing 1% Avicel in a ratio 1:1 was applied to infected monolayers of MDCK-Atlanta cells that were subsequently cultured at 37°C in a humidified atmosphere 5% CO₂ for 72 hours. Cells were then washed with 1X PBS three times and stained with crystal violet, and viral plaques were counted. Viral titers were calculated and presented as lung viral titer (PFU, Log₁₀).

WT and Rgs10-/- mice were sacrificed as indicated above, and murine lungs were intratracheally inflated with 1 ml of 10% Neutral Buffered Formalin. The trachea was tightened, and lungs were collected into 10% Neutral Buffered Formalin. Formalin-fixed/paraffin-embedded lung tissue blocks were prepared by the Histology Laboratory at the College of Veterinary Medicine at UGA and were sectioned at 5 µm thickness. Lung tissue sections were either left unstained for subsequent immunofluorescence experiments or stained with hematoxylin and eosin (H&E). H&E slides were analyzed by a pathologist who was blinded to the experiments. The lung histological score was determined on a scale from 0 to 4 based on a combination of assessments including bronchioles and alveolar space structure and infiltration of inflammatory cells and aggregation in alveoli, bronchioles, interstitium, blood vessels and pleura. A score of 0 represented no damage; 1 represented mild damage; 2 represented moderate damage, 3 represented severe damage; and 4 represented very severe damage and marked histological changes as done previously (25).

Lung tissue sections were deparaffinized, and rehydrated with xylene and alcohol gradient. Sections were antigen-retrieved with 0.1 M sodium citrate in Pascal Pressure Cooker (DakoCytomation, Aligent Technologies, Santa Clara, CA) for 20 min at 95 °C and then permeabilized with 0.1% Triton X100 while blocking in PBS with 5% BSA and 10% normal horse serum for 1 h. Primary antibody staining to detect Influenza A Virus Nucleoprotein (NP) (BEI Resources, Cat# NR-43899) was performed at a 1:250 dilution in PBS with 1% BSA, 1% normal horse serum, and 0.3% Triton X100 overnight at 4 °C. Sections were washed in 1X-TPBT, and secondary antibody staining was performed at a 1:1,000 dilution in PBS with 1% BSA, 1% normal horse serum for 1 h with horse anti-mouse IgG antibody (H+L), fluorescein (Vector Laboratories, cat#: FI-2000-1.5). Sections were washed with 1X-TPBS and then vectashield™ anti-fade mounting medium with DAPI (Vector Laboratories, cat#: H-1200-10) was applied to the lung tissue sections prior coverslip addition. All digital images were acquired at the University of Georgia College of Veterinary Medicine Cytometry Core on a Nikon A1R confocal microscope (Nikon Eclipse Ti-E inverted microscope) and examined with NIS Element software (Nikon, Version 6.4).

Total RNA was isolated from lung homogenate using TRIzol reagent (Invitrogen, Cat#: 15596018). cDNA was synthesized from 2 μg of total RNA using the High-capacity Reverse Transcriptase cDNA kit (Applied Biosystem, Cat#: 4368814). Each cDNA sample was diluted 10-fold, and 5 µl was used in a 15 µl PCR reaction (SYBR™ Green PCR Master Mix.) (Thermo Fisher Scientific, cat#: 4309155) containing primers at concentration of 5 µM each. All the reactions were run in triplicates. The mRNA expression levels were normalized to the housekeeping β-actin gene and were calculated using the 2− ^ΔΔCT method. Mouse Rgs2, Rgs10, Rgs12, Rgs14 and β-actin primers were obtained from Millipore Sigma. The primer sequences used for gene amplification are listed as follows: Rgs2 forward, 5′- GAGAAAATGAAGCGGACACTCT-3’, Rgs2 reverse, 5′- GCAGCCAGCCCATATTTACTG-3’, Rgs10 forward, 5’-CCCGGAGAATCTTCTGGAAGACC-3’, Rgs10 reverse, 5’-CTGCTTCCTGTCCTCCGTTTTC-3’, Rgs12 forward, 5′- GTGACCGTTGATGCTTTCG-3’, Rgs12 reverse, 5′- ATCGCATGTCCCACTATTCC-3’, Rgs14 forward, 5′- AAATCCCCGCTGTACCAAG-3’, Rgs14 reverse, 5′- GTGACTTCCCAGGCTTCAG-3’, Actb forward, 5′- GGCTGTATTCCCCTCCATCG-3’, Actb reverse, 5′-CCAGTTGGTAACAATGCCATGT-3’.

MPO-DNA complexes were detected in the BALF of infected WT and Rgs10-/- mice following a published protocol (26, 27) adapted to murine samples (28). BALFs were diluted 1:25 in PBS and were subsequently added to a high-binding 96-well ELISA microplate pretreated overnight at 4 °C with capture anti-MPO (1:200 RD Systems, Cat#: AF3667). The wells of microplate were blocked with 5% BSA in PBS at room temperature for 2 hours. Samples were incubated overnight at 4 °C. Following three washes with PBS/Tween-20, the secondary mouse anti-DNA-POD (HRP-conjugated anti-DNA antibody, 1:500, Roche, Cat#: 1154467501) was added for 1 h at room temperature. Samples were washed four times with PBS/Tween 20. TMB substrate (Thermo Scientific, Cat#34021) was added, and the reaction was stopped by the addition of 1 M HCl. Absorbance was measured at 450 nm with an Eon microplate spectrophotometer (BioTek, Winooski, VT). Background absorbance values of the medium were subtracted. All samples being compared were run in duplicates on the same plate. Differences between optical densities were compared.

Neutrophil elastase (NE) enzymatic activity of the IAV-infected BALFs was determined using the NE activity assay kit that uses a specific NE substrate (Z-Ala-Ala-Ala-Ala) 2Rh110 (Cayman Chemical, cat#: 600610). The NE enzymatic activity in BALFs was determined by measuring NE-dependent cleavage of 2Rh110 to highly fluorescent compound R110. This fluorescence intensity was read using an excitation of 485 nm and emission wavelength of 525 nm. The standard curve was generated using human NE at 18 U/ml and were serial diluted from 10 mU/ml - 0 mU/ml in assay buffer provided with the kit. The substrate was added to each well and put in the Varioskan Flash™ microplate fluorimeter (Thermo Fisher Scientific, Waltham, MA). The plate was then incubated at 37°C throughout the fluorescence readings and read for 1.5 hours. Fluorescence measurements were taken every 2 minutes for 1.5 hours, and the average fluorescence for each well was measured. To calculate the NE concentration in each sample in mU/ml, the average fluorescence for each standard was calculated, and the blank was subtracted from each well. Based on the standard curve generated: NE (mU/ml) = [(fluorescence – (y-intercept))/slope] x dilution factor.

All quantified data were analyzed for statistical difference between groups using unpaired student t-test (for comparing two groups) or one-way ANOVA followed by Tukey post-hoc analysis (for comparing three or more groups). GraphPad Prism software was used to carry out statistical analyses. Data are expressed as mean ± S.E.M. where *, p<0.05; **, p<0.01; and ***, p<0.001 indicate the levels of significance. The relevant statistical information including n values and p-values are given in individual figure legends.

While previous studies have investigated the impact of RGS10 on the pathophysiology of diverse disease models using Rgs10-/- mice (13), no work has studied the in vivo role of RGS10 in fighting infections. To explore the in vivo role of RGS10 in response to viral challenge, eight-to-ten-week-old, sex- and weight-matched Rgs10-/- mice and their Rgs10-expressing C57BL/6 wild-type (WT) littermate control mice were infected intranasally (i.n.) with a lethal dose of 100 plaque-forming units (PFU) of the mouse-adapted influenza A/Puerto Rico/8/1934 H1N1 (PR8) virus. While most people infected with influenza do not die [its mortality rate is around 0.1% (1)], we decided to study the role of RGS10 in a murine model of lethal influenza infection because severe infections are associated with worse clinical outcomes requiring hospitalization and expensive treatments, mainly in immunocompromised patients. Survival and body weight loss were monitored for eight days. Rgs10-/- mice demonstrated significantly increased mortality compared to WT animals ( Figure 1A ). Both WT and Rgs10-/- mice started losing body weight gradually from day 3 post-infection ( Figure 1B ), but Rgs10-/- mice lost significantly more body weight compared to WT mice (3 through 6-day post-infection, dpi) ( Figure 1C ). In addition to prior published data using western blotting (13), the absence of Rgs10 expression was also confirmed in this study using qRT-PCR in lungs extracted from Rgs10-/- mice ( Figure 1D ). No effect of Rgs10 deficiency was observed on the transcript expression of Rgs2, the widely expressed RGS protein with high expression in airways, or on the transcript levels of Rgs12 and Rgs14, which share high sequence identity with the RGS domain of RGS10 protein ( Figure 1D ). These results indicate that RGS10 delays the onset of the clinical consequences of lethal respiratory IAV infection.

Next, we investigated whether the increased weight loss and death resulting from IAV infection in Rgs10-/- mice were associated with more severe lung damage. To test this, we inoculated WT and Rgs10-/- mice i.n. with 100 PFU influenza virus, and 3 and 5 dpi, we assessed the pathological changes in the lung tissues. We chose these time points (3 and 5 dpi) to assess lung injury because 3 dpi showed the earliest difference in weight loss between genotypes, and 5 dpi showed the earliest and largest difference in survival between strains. Approximately 42.3% of Rgs10-/- mice died at 5 dpi (survival rate, 57.7%) in response to 100 PFU PR8 infection compared to 11.5% of mortality rate in infected WT mice. Following IACUC guidelines and the approved protocol at UGA, mice that lost greater than 20% of their initial weight at day of infection were considered moribund and subsequently removed from the ongoing experiment. To examine the lung histopathology of uninfected animals or infected mice at 3 and 5 dpi, lung sections collected from WT and Rgs10-/- mice stained with hematoxylin and eosin (H&E). No histopathological differences were observed in the lungs between uninfected (0 dpi) WT and Rgs10-/- mice ( Figure 2A ). Despite the significant weight loss in infected Rgs10-/- mice, no differences in the signs of lung damage were observed between WT and Rgs10-/- mice at 3 dpi ( Supplementary Figures S1A, B ). More importantly, at 5 dpi, Rgs10-/- mice developed severe lung damage demonstrated by massive inflammatory cell infiltration, such as neutrophils, into the bronchioles (bronchiolitis) ( Figures 2A, B ) and inflammatory cells aggregation into the alveolar walls and spaces (alveolitis) ( Figures 2A, C ). Unlike Rgs10-/- mice, infected WT mice only showed minimal bronchial cell infiltrates and alveolar cell aggregates ( Figures 2A – C ). To further assess lung damage, we measured the total protein concentration in BALFs, as an indicator of general inflammation and epithelial leakage. There was no significant difference in the BALF protein levels between WT and Rgs10-/- mice at 0 dpi ( Supplementary Figure S1C ). However, the results showed that protein concentrations were significantly higher in the BALFs of PR8-infected Rgs10-/- mice at 3 dpi (1,152 ± 280.2 μg/ml) ( Supplementary Figure S1C ) and at 5 dpi (2,019 ± 144.1 μg/ml) ( Figure 2D ) compared to their WT control counterparts (826.9 ± 154.2 μg/ml) ( Supplementary Figure S1C ) and (1,339 ± 228.6 μg/ml) ( Figure 2D ), respectively. Altogether, the absence of Rgs10 is associated with increased tissue damage, bronchiolitis, alveolitis and general inflammation in influenza-infected mice.

To determine if the increased inflammatory response-mediated lung injury of Rgs10-/- mice correlated with higher viral loads, we measured lung virus titers using the plaque assay. WT and Rgs10-/- mice were infected as described above. At 5 dpi, infected lungs were collected and processed to determine the lung viral titers using MDCK-Atlanta cells. Viral loads in Rgs10-/- mouse lungs were significantly higher (1.4 log difference) than the viral loads in WT mouse lungs (P=0.0097) ( Figure 3A ). To further confirm that Rgs10-/- mouse lungs retain higher viral loads following IAV infection, sectioned lungs from PR8-infected WT and Rgs10-/- mice at 5 dpi were probed with anti-IAV nucleoprotein (NP) antibody. The results confirmed increased viral loads in the lungs of Rgs10-/- mice compared to WT littermate controls ( Figure 3B ). Overall, these findings indicate that lack of RGS10 results in elevated viral titers in the lungs following IAV infection.

Dysregulation of the inflammatory response is a major factor by which IAV infection causes morbidity and mortality (29). Because of the increased protein concentrations and viral loads in Rgs10-/- airways, we next quantified cytokine and chemokine levels in BALF collected from uninfected (0 dpi) or PR8-infected WT and Rgs10-/- mice. In general, there were no observed differences in the BALF levels of any cytokines and chemokines between uninfected (0 dpi) WT and Rgs10-/- mice ( Figure 4 and Supplementary Figure S2 ). At 5 dpi, Rgs10-/- mice demonstrated considerably higher levels of certain cytokines and chemokines including IL-1β, IL-6, CCL1, MCP-1 (CCL2), CCL3, CCL4, CCL5, CCL11, CCL12, CCL20, KC (CXCL1), CXCL10, and CXCL13, respectively ( Figure 4 ). There were elevated, but not significantly different levels of GM-CSF, IFN-γ, CCL7, CCL19, CCL22, and CCL24 ( Supplementary Figure S2 ), and no differences were observed in several other cytokine and chemokine levels ( Supplementary Figure S2 ). Thus, these data suggest that deficiency of RGS10 is implicated in shaping the immune response by enhancing the release of inflammatory cytokines and chemokines in infected mouse lungs.

To determine whether the increased levels of cytokines and chemokines manifested in increased numbers of particular leukocyte subsets, we characterized the composition of infiltrating and residential immune cells in the BALF of both uninfected or PR8-infected WT and Rgs10-/- mice at 3 and 5 dpi. Flow cytometry was performed using surface markers to distinguish and determine the numbers of myeloid (neutrophils, monocytes, inflammatory monocytes, eosinophils, dendritic cells, alveolar (pulmonary) macrophages and inflammatory (monocyte-derived) macrophages) ( Supplementary Figure S3 ) and lymphoid (T cells, B cells, and natural killer (NK) cells) ( Supplementary Figure S4 ) cell subsets. As expected, alveolar macrophages represented the dominant immune cell type in uninfected airways ( Supplementary Figure S5A ). There were no differences in the number of CD11b⁺ leukocytes or in the number of any subset of myeloid and lymphoid cells between mouse strains without infection ( Supplementary Figure S5 ) or at 3 dpi ( Supplementary Figure S6 ). However, at 5 dpi, the total number of CD11b⁺ cells was about two times higher in Rgs10-/- mice compared to WT animals ( Figure 5 ). This significant increase in total CD11b⁺ leukocyte counts was most likely accounted for by significantly higher numbers of neutrophils, monocytes and inflammatory monocytes in PR8-infected Rgs10-/- mice in comparison to infected WT animals ( Figure 5 ). Analysis of BALF from WT and Rgs10-/- mice at 5 dpi exhibited no statistically significant differences in the number of eosinophils, dendritic cells, alveolar macrophages, inflammatory macrophages, T cells, B cells and NK cells ( Figure 5 ). Collectively, the increased susceptibility to IAV challenge in Rgs10-/- mice is associated with excessive influx of neutrophils and monocytes to the lungs.

Neutrophils have previously been shown to have a protective role following influenza virus infection (30). However, growing evidence strongly implicates that accumulation of neutrophils following severe infections can excessively release NETs, which trigger inflammatory responses and result in tissue injury-mediated organ dysfunction (31). Because of the abundant neutrophil infiltrates in infected airways of Rgs10-/- mice, we explored whether soluble markers of NETs are also increased in the lungs of PR8-infected Rgs10-/- mice. Cell-free BALF supernatants were collected from WT and Rgs10-/- mice at 0 and 5 dpi and the presence of NET-specific MPO-DNA complexes was assessed as previously (26–28). While there was no significant difference in the BALF MPO-DNA complexes level between WT and Rgs10-/- mice at 0 dpi, we found that there was a significant, 4-fold increase in the absorbance values in the BALFs of infected Rgs10-/- mice compared to their WT littermate controls ( Figure 6A ), indicating the enhanced release of NETs in infected airways of these animals. To further characterize the consequences of neutrophils’ presence in infected Rgs10-/- lungs, we measured NE enzymatic activity, one of the key enzymes in neutrophils and NETs (32). Our data revealed that PR8-infected Rgs10-/- lungs have significantly higher BALF NE activity (7.2 mU/ml) than their WT control groups (2.9 mU/ml) ( Figure 6B ). Therefore, the above results demonstrate more NETs and NE released in the airways of Rgs10-/- mice that likely contribute to the observed tissue damage in the lung.