key: cord-0853030-bt29t64w
authors: Tian, Jide; Middleton, Blake; Kaufman, Daniel L.
title: GABA administration limits viral replication and pneumonitis in a mouse model of COVID-19
date: 2021-02-16
journal: bioRxiv
DOI: 10.1101/2021.02.09.430446
sha: 38c2c5a37e4b38c19e85a199e69c99a9fcd0c4d6
doc_id: 853030
cord_uid: bt29t64w

Despite the availability of vaccines for COVID-19, serious illness and death induced by coronavirus infection will remain a global health burden because of vaccination hesitancy, possible virus mutations, and the appearance of novel coronaviruses. Accordingly, there is a need for new approaches to limit severe illness stemming from coronavirus infections. Cells of the immune system and lung epithelia express receptors for GABA (GABA-Rs), a widely used neurotransmitter within the CNS. GABA-R agonists have anti-inflammatory effects and can limit acute lung injury. We previously showed that GABA treatment effectively reduced disease severity and death rates in mice following infection with a coronavirus (MHV-1) which provides a potentially lethal model of COVID-19. Here, we report that GABA treatment also reduced viral load in the lungs, suggesting that GABA-Rs may provide a new druggable target to limit pulmonary coronavirus replication. Histopathological analysis revealed that GABA treatment reduced lung inflammatory infiltrates and damages. Since GABA is safe for human consumption, inexpensive, and available worldwide, it is a promising candidate to help treat COVID-19.

While GABA-Rs are well known for their role in neurotransmission in the CNS, these receptors are also found on some cells in the periphery, most notably for our studies, on some cells of the immune system and lung epithelia. The biological roles of GABA-Rs on immune cells are not yet well understood, but there is a growing body of evidence that the activation of these receptors has immunoregulatory actions. Rodent and human macrophages and dendric cells express GABA-Rs and GABA-R agonists inhibit their inflammatory activities (1) (2) (3) (4) (5) (6) . T cells also express GABA-Rs (4) (5) (6) (7) (8) . The administration of GABA-R agonists inhibits autoreactive Th1 and Th17 cells while promoting CD4 + and CD8 + Treg responses (5, 6, 9) and ameliorates autoimmune disease in mouse models of type 1 diabetes (T1D), multiple sclerosis, and rheumatoid arthritis, and also limits inflammation in murine type 2 diabetes (1, 5, 6, 10, 11) . Human immune cells also express GABA-Rs and GABA inhibits secretion of IL-6, TNF, IL-17A, CXCL10/IP-10, CCL4, CCL20, and MCP-3 from anti-CD3 stimulated PBMC from T1D patients (7) . The ability of GABA-R agonists to inhibit the production of number of inflammatory factors is of potential interest for helping to treat COVID-19 since high levels of some of these inflammatory factors in patient sera is associated with the development of severe COVID-19 (12) (13) (14) (15) .

Lung epithelial cells also express GABA-Rs, specifically the GABAA-R subtype.

GABA and GABAA-R positive allosteric modulators (PAMs) have been shown to reduce inflammation and improve alveolar fluid clearance and lung functional recovery in different rodent models of acute lung injury (16) (17) (18) (19) (20) (21) (22) (23) , as well as limit pulmonary inflammatory responses and improve clinical outcomes in ventilated human patients (24) (25) (26) . GABAA-R PAMs reduce macrophage infiltrates and inflammatory cytokine levels in bronchoalveolar lavage fluid (BALF) and limit inflammatory responses by rodent and human macrophages (3, 22, (27) (28) (29) (30) (31) . GABA application reduces the secretion of inflammatory factors from LPS-stimulated human bronchial epithelial cells in vitro (17) . Finally, GABA can inhibit platelet aggregation (32) , which is important because pulmonary thrombosis often occurs in critically ill COVID-19 patients (33, 34) .

used as a safe model of SARS-CoV and SARS-CoV-2 infection (35) (36) (37) (38) . Intranasal inoculation with ³5 x 10 3 plaque-forming units (PFU) of MHV-1 in A/J mice induces acute pneumonitis and acute respiratory distress syndrome with a high lethality rate.

The MHV-1-infected mice develop clinical symptoms and pathological features similar to those in severely ill COVID-19 patients, including high levels of pulmonary edema, pneumonitis, dense macrophage infiltrates, hyaline membranes, fibrin deposits, accompanied by loss of body weight and respiratory distress (35) (36) (37) (38) . We previously showed that GABA treatment just after MHV-1 inoculation, or after the appearance of disease symptoms, very effectively protected the mice from severe illness and death (39) . Here, we report that GABA treatment also reduced viral loads and pathological findings in their lungs. We discuss possible mechanisms underlying these observations.

Mice. Female A/J mice (8 weeks in age) were purchased from the Jackson Laboratory and maintained in microisolator cages and fed with a standard diet and water ad libitum.

This study was carried out in accordance with the recommendations of the Guide for the Finally, the pneumonitis score of individual mice = (% of inflammation areas x severity score) + lung consolidation score + hyaline membrane score with a maximum score of 11.

A/J mice were inoculated intranasally with MHV-1 (5 x 10 3 PFU) and then randomized to receive plain water (controls) or water containing GABA (20 mg/mL, as in (39) ) for the remainder of the study. Mice from these groups were euthanized 3 or 6 days post-infection and the virial load in their lungs was determined. Concurrently, another group of MHV-1 inoculated mice was given water containing GABA beginning at 3 days post-infection and the viral load in their lungs was determined 6 days postinfection.

At three days post-infection the mean viral load in the lungs of mice given plain water was about 7-fold higher than that in mice given GABA immediately after infection (p<0.5, Fig. 1) . Thereafter, the viral load in the lungs declined as expected, and by day 6 post-infection the viral load in the lungs of control mice were about twice that in the lungs of mice given GABA either immediately or 3 days post-infection, although these differences were not statistically significant (Fig. 1) . Thus, early GABA treatment reduced viral loads in the lungs of mice.

Histological evaluation of lung sections from mice given GABA immediately following MHV-1 inoculation revealed reduced inflammatory infiltrates, hyaline-like membrane formation and fibrin deposits in the alveoli at 3 days post-infection, relative to that in control MHV-1 inoculated mice ( Fig. 2A) . Thus, GABA treatment limited the MHV-1 induced lung damage in A/J mice.

Our previous study showed that GABA treatment beginning just after MHV-1 inoculation or after the appearance of symptoms rapidly curtailed disease progression (39) . GABA-treated mice also had smaller lung coefficient indexes (indicative of less inflammation and edema). Thus, GABA-R activation can limit a very acute and highly lethal viral infection-induced pulmonary inflammation, a property that heretofore was unknown. Here, we show that early GABA treatment reduced MHV-1 replication in the lungs of mice when measured near the time of peak viral load in this model (35) (36) (37) , which may have contributed to the better outcomes observed in GABA-treated animals.

Hence, GABA-R agonists may provide a new approach to limit viral replication in the lungs.

We can envision a number of ways that GABA may have limited viral replication, including: 1) The lung alveolar cells of rodents and humans express GABAA-Rs (30, 42) .

While the activation of GABAA-R's Clchannels on neurons leads to Cl-influx and hyperpolarization, the activation of GABAA-Rs on ATII cells induces Clefflux and greater membrane depolarization (30, 42) . Because coronaviruses promote Ca 2+ influx to enhance their replication (43, 44) , the activation of ATII GABAA-Rs and the ensuing Clefflux and membrane depolarization may limit the influx of extracellular Ca 2+ , making the cellular environment less conducive to viral replication. 2) Activation of GABAA-Rs on lung alveolar and large airway epithelial cells may have A) altered the secretion of immune signaling molecules from infected cells, B) altered alveolar surfactant production/absorption, and/or C) altered inflammatory responses and autophagy (45) and, D) reduced the expression of the MHV-1 receptor CAECAM1 in ways that limited virus production. Further detailed studies are needed to evaluate whether these factors, and/or others, contributed to the observed reduction in viral load.

Histopathological analysis of lungs from mice that received GABA just after MHV-1 inoculation revealed that three days post-infection these mice had significantly reduced lung inflammation and markers of lung damage relative to that in control mice.

There are a number of different biological processes through which GABA treatment may have ameliorated the severity of pneumonitis: 1) GABA can inhibit macrophage and dendritic cell inflammatory activities (1-3, 29, 46, 47) . Likewise, GABAA-R PAMs reduce the numbers of macrophages in broncholavage fluid, lung secretion of inflammatory cytokines, and inflammatory responses by rodent and human macrophages (22, (27) (28) (29) (30) (31) . GABAA-R agonists also inhibit activated Th17 and Th1 responses and promote CD4 + and CD8 + Tregs, however, since adaptive immune responses take time to arise, these abilities are unlikely to have contributed to GABA's ability to attenuate disease soon after MHV-1 infection. These effects on adaptive immune responses may be relevant for treating COVID-19 which has a longer disease course and in which high levels of circulating Th1, Th17, and Th2-secreted proteins are associated with severe illness (12, 13) . In murine models of pneumatic bacterial infections, GABAA-R agonists reduced bacterial load and TNFa and IL-6 levels in the lungs and protected the mice against illness (45) . 5) GABA inhibits platelet aggregation (32)-this may be an important property because pulmonary thrombosis is increased in critically ill COVID-19 patients (33, 34) . Thus, treatment with GABA may have led to better outcomes in MHV-1 infected mice through multiple and diverse pathways.

Much remains to be learned about the mechanisms by which GABA-R activation protected MHV-1 infected mice from severe pneumonitis and whether these observations extend to SARS-CoV-2 infections. Given that GABA can affect many biological processes and that viral infection is a very dynamic process it is clear that GABA-R agonist dosing needs to be carefully studied and optimized for different stages of coronavirus infection. Our observations provide a spring board for future investigations into whether the GABA system can be modulated to limit severe illness due to SARS-CoV-2, future novel coronavirus outbreaks, and other respiratory disorders.

Oral GABA treatment downregulates inflammatory responses in a mouse model of rheumatoid arthritis

Inhibitory role for GABA in autoimmune inflammation

Autocrine GABA signaling distinctively regulates phenotypic activation of mouse pulmonary macrophages

GABA protects human islet cells against the deleterious effects of immunosuppressive drugs and exerts immunoinhibitory effects alone

Homotaurine treatment enhances CD4+ and CD8+ Treg responses and synergizes with low-dose anti-CD3 to enhance diabetes remission in type 1 diabetic mice

Homotaurine, a safe bloodbrain barrier permeable GABAA-R-specific agonist, ameliorates disease in mouse models of multiple sclerosis

GABA Regulates Release of Inflammatory Cytokines From Peripheral Blood Mononuclear Cells and CD4(+) T Cells and Is Immunosuppressive in Type 1

GABA(A) receptors mediate inhibition of T cell responses

Combined therapy with GABA and proinsulin/alum acts synergistically to restore long-term normoglycemia by modulating T-cell autoimmunity and promoting beta-cell replication in newly diabetic NOD mice

Gamma-aminobutyric acid inhibits T cell autoimmunity and the development of inflammatory responses in a mouse type 1 diabetes model

Oral treatment with gamma-aminobutyric acid improves glucose tolerance and insulin sensitivity by inhibiting inflammation in high fat diet-fed mice

Sinai Immunology Review P. Immunology of COVID-19: Current State of the Science

Longitudinal analyses reveal immunological misfiring in severe COVID-19

Pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology

Lessons for COVID-19 Immunity from Other Coronavirus Infections

Propofol reduces acute lung injury by upregulating gamma-aminobutyric acid type a receptors

Effects of anesthetic regimes on inflammatory responses in a rat model of acute lung injury

GABA receptor ameliorates ventilator-induced lung injury in rats by improving alveolar fluid clearance

Baclofen, a GABABR agonist, ameliorates immune-complex mediated acute lung injury by modulating proinflammatory mediators

Sevoflurane ameliorates gas exchange and attenuates lung damage in experimental lipopolysaccharide-induced lung injury

The volatile anesthetic isoflurane prevents ventilator-induced lung injury via phosphoinositide 3-kinase/Akt signaling in mice

Effects of propofol on hemodynamic and inflammatory responses to endotoxemia in rats

Desflurane Attenuates Ventilator-Induced Lung Injury in Rats with Acute Respiratory Distress Syndrome

Immunomodulatory Effects of Anesthetics during

Thoracic Surgery

Anesthetic-induced improvement of the inflammatory response to one-lung ventilation

Effects of propofol and desflurane anaesthesia on the alveolar inflammatory response to one-lung ventilation

Effect of Propofol on the Production of Inflammatory Cytokines by Human Polarized Macrophages

A Novel Orally Available Asthma Drug Candidate That Reduces Smooth Muscle Constriction and Inflammation by Targeting GABAA Receptors in the Lung

Anaesthetic impairment of immune function is mediated via GABA(A) receptors

Isoflurane regulates atypical type-A gamma-aminobutyric acid receptors in alveolar type II epithelial cells

Sevoflurane and isoflurane decrease TNF-alpha-induced gene expression in human monocytic THP-1 cells: potential role of intracellular IkappaBalpha regulation

Characteristics of endogenous gamma-aminobutyric acid (GABA) in human platelets: functional studies of a novel collagen glycoprotein VI inhibitor

Platelet gene expression and function in patients with COVID-19

Platelet activation and platelet-monocyte aggregate formation trigger tissue factor expression in patients with severe COVID-19

Murine hepatitis virus strain 1 produces a clinically relevant model of severe acute respiratory syndrome in A/J mice

Protective and pathologic roles of the immune response to mouse hepatitis virus type 1: implications for severe acute respiratory syndrome

Toll-like receptor 4 deficiency increases disease and mortality after mouse hepatitis virus type 1 infection of susceptible C3H mice

T cell epitope specificity and pathogenesis of mouse hepatitis virus-1-induced disease in susceptible and resistant hosts

GABA administration prevents severe illness and death following coronavirus infection in mice

Coronaviruses: propagation, quantification, storage, and construction of recombinant mouse hepatitis virus

Trimmed Spearman-Karber method for estimating median lethal concentrations in toxicity bioassays

A novel function of ionotropic gammaaminobutyric acid receptors involving alveolar fluid homeostasis

Porcine deltacoronavirus (PDCoV) modulates calcium influx to favor viral replication

Mouse hepatitis virus infection induces an early, transient calcium influx in mouse astrocytoma cells

GABAergic signaling linked to autophagy enhances host protection against intracellular bacterial infections

The anti-spasticity drug baclofen alleviates collagen-induced arthritis and regulates dendritic cells

Antiinflammatory effects of the GABA(B) receptor agonist baclofen in allergic contact dermatitis

to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors approved the final manuscript as submitted.