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Copyright © 2020 by the Shock Society. Unauthorized reproduction of this article is prohibited. Copyright © 2020 by the Shock Society. Unauthorized reproduction of this article , NY es [email protected] [email protected] 10.1097/SHK.0000000000001610 mail: mail: mail: mail: - - Address Department of Surgery, Zucker School of Medicine at Hofstra/Northwell, Hofstra/Northwell, Medicine at of School Zucker Surgery, of Department Center for Immunology and Inflammation, The Feinstein Institutes for Medical Research, Medical Research, for Feinstein The Institutes and Inflammation, Immunology for Center Zucker Medicine, Molecular of Department

Assistant Professor Assistant and Inflammation Immunology for Center Research for Medical Institutes Feinstein The Ma Dr., 350 Community E 350 Community Dr., Manhasset, NY 11030 NY Manhasset, Dr., 350 Community 562 (516) Tel: 562 (516) Fax: E PhD Aziz, Monowar * MD Wang, Ping (CSO) Scientific Officer Chief and Professor Research for Medical Institutes Feinstein The non financial and/or Competing interests. competing Manhasset title: Running Manhasset, NY Manhasset, 3 1 2 SHOCK: Publish Ahead Print Ahead Publish SHOCK: DOI:

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Coronavirus disease 2019 (COVID-19) is a life-threatening respiratory illnesscaused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).Its clinical presentation can vary from the asymptomatic state to acute respiratory distress syndrome (ARDS) and multi- organ dysfunction. Due to ourinsufficient understanding of its pathophysiology and lack of effective treatment,the morbidity and mortality ofsevere COVID-19 patients are high. Patients with COVID-19developARDSfueled byexaggerated neutrophil influx into the lungs and cytokine storm. B-1a cells represent a unique subpopulation of B lymphocytescritical for circulating natural antibodies, innate immunity,andimmunoregulation. These cells spontaneously produce natural IgM, interleukin (IL)-10, and granulocyte-monocytecolony stimulating factor

(GM-CSF). Natural IgM neutralizes viruses and opsonizes bacteria, IL-10 attenuates the cytokine storm, and GM-CSF induces IgM production by B-1a cells in an autocrine manner.

Indeed, B-1a cells have been shown to ameliorate influenza virus infection,sepsis, and pneumonia, all of which are similar to COVID-19. The recentdiscovery of B-1a cells in humans further reinforces their potentially critical role in the immune response against SARS-CoV-2 and their anticipated translational applications againstviral and microbial infections. Given that B-1a cells protect againstARDS via immunoglobulin production and theanti-COVID-19 effects ofconvalescent plasmatreatment, we recommend that studies be conducted to further examine the role of B-1a cells in the pathogenesis of COVID-19 and explore theirtherapeutic potential to treat

COVID-19 patients.

Key words: COVID-19; SARS-CoV-2; B-1a cells; IL-10; IgM; Neutrophils; ARDS;Sepsis

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by therecently identified and highly contagious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-

2)(1). COVID-19 predominantly affectsthe respiratory systembecause the virus binds to cells expressingthe angiotensin-converting enzyme 2 (ACE2), which is abundantin the surface of type

II alveolar cells of the lungs(2, 3). The virus uses itstransmembrane spike (S) glycoprotein to connect to ACE2 and penetrate the host cellwhere ithijacksthe cell machineryto multiply (2,

3).Many patients are asymptomatic or show only flu-like symptoms, but a substantial subset of

COVID-19 patients develop severe pneumonia that often progresses toacute respiratory distress syndrome (ARDS)fueled by excessive neutrophilinfiltration and uncontrolled production of pro- inflammatory cytokines in the lungs(4-7).

At the moment, the treatmentfor patients with COVID-19 is essentially supportive and symptomatic. There is a long list of drugs being evaluated and clinical trials being carried out(8), but none of them has yet proven to be a game changerfor patients withCOVID-19.Promising treatments include monoclonal antibodies against pro-inflammatory cytokines (8), immunoglobulin therapy,andantibody-containing convalescent plasmafrom recovered patients (8,

9).

B-1a cells consist of a unique subset of B lymphocytes which play an early and key role in the innate immune response against viral, bacterial, and acute inflammatory diseases(10).

These cells release polyreactivenatural IgM which non-specifically recognizesand neutralizes microbes(10-12). These cells also spontaneously produce the anti-inflammatory cytokines interleukin (IL)-10 and IL-35 and the immune bolsteringfactor granulocyte-monocyte colony stimulating factor (GM-CSF)(10, 13-15).B-1acells andsubpopulationswere originally described

Copyright © 2020 by the Shock Society. Unauthorized reproduction of this article is prohibited. in the mouse and have also beenrecently identified in humans (16). B-1a cells ameliorate acute viral and bacterial infections in rodent models of influenza, polymicrobial sepsis, and pneumonia(10). B-1a cell therapyis able to mitigate the progression of these infections to

ARDS,raising the possibility that B-1a cell therapymay alsoameliorate ARDS associated with

COVID-19.

Here, we review the protective effects ofB-1a cells in animal models of ARDScaused by viral and bacterialinfections with the caveat that sepsis and influenza may not accurately reflect the immune processes of COVID-19. We compare the pathophysiological and clinical aspects between bacterial sepsis and severe COVID-19 in Tables 1 and 2. Weanalyze the literature on B-

1a cells with a focus on the excessive neutrophil infiltration, abnormal neutrophil function, and the cytokine storm which have been described in numerous studies of patients with COVID-19.

By comparing the existing B-1a cell literature and recent advances in the pathogenesis of

COVID-19, we propose a novel perspective suggesting that treatment with B-1a cells may benefit patients with COVID-19.Since many clinical trials in sepsis have failed and almost no drug has yet been developed to treat sepsis patients,we acknowledge an obstacle of implementation of B-1a cells as a therapeutic candidate in COVID-19 because the beneficial outcomes of B-1a cells as reported here were mainly built upon sepsis data.For designing and conducting immunotherapies for COVID-19, the lessons learned from the sepsis clinical trials should be reminisced(17).

COVID-19complications and pathophysiology

While many infected individuals develop mild or moderate symptoms, a significant fraction of patients develop severe pneumonia, ARDS, multi-organ failure, and septic shock, not rarely resulting in death(4, 18). Cardiovascular complications may include heart failure, arrhythmias,

Copyright © 2020 by the Shock Society. Unauthorized reproduction of this article is prohibited. myocarditis, and hypercoagulability(19). In addition, COVID-19 patients may sometimes develop liver injuryand acute kidney injury (20, 21). Its related neurologic manifestations include seizure, stroke, and encephalitis(22). The elderly are particularly vulnerable to severe SARS-

CoV-2 infection, with a mortality rate higher than that of younger individuals, which may be due to a combination of immune senescence and the presence of co-morbidities(23, 24).However, following SARS-CoV-2 infection, patients of all ages can develop COVID-19, including children who sometimes developa severe systemic inflammation that can be lethal (25).

SARS-CoV-2is a membrane-enveloped positive sense single-stranded RNAvirus of 50-

200 nm in diameter. It has four structural proteins: thespike(S), envelope (E), membrane (M), and nucleocapsid (N) proteins(26). The S protein has strong affinity for ACE2 on human cells,which it uses asa receptor to attach to and penetrate host cells, where it multiplies intonumerous copies(2).The higher expression of ACE2 in lung, heart, ileum, and kidney cells explains why these organs are affected more than others (26, 27).

After intracellular replication and maturation, the host cell bursts releasing the virionsand starting a new cycle of infection(26). As cells rupture (lytic release), it is speculated that they also release intracellular components, such as extracellular cold-inducible RNA binding protein

(eCIRP), double-stranded DNA, histones, and high mobility group box 1 (HMGB1)(28). Nearby cells get activated by the viral single-stranded RNA and proteins via pathogen-associated molecular pattern (PAMP) receptors, as well as by the ruptured cell contents viadamage- associated molecular pattern (DAMP) receptors(28, 29). Downstream signaling from these receptors leads to the release chemokines, antiviral cytokines, and pro-inflammatory cytokines and other mediators. As a result, patients with severe COVID-19 havea cytokine storm characterized byelevated levels of IL-2, IL-7, IL-6, tumor necrosis factor-α (TNF-α), interferon-γ

Copyright © 2020 by the Shock Society. Unauthorized reproduction of this article is prohibited. inducible protein 10, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1-α(4, 30).Additionally, COVID-19 patients with ARDS have serum biomarkers of systemic inflammation such as elevated like C-reactive protein, lactate dehydrogenase, D- dimers, and ferritin(4).

Systemicinflammation results in vasodilation and chemotaxis, leading to the pro- inflammatory lymphocytic and monocytic infiltration of the lungs and heart, which further fuel the cytokine storm(31). Peripheral and lung innate immune responses to severe SARS-CoV-2 infection as well asthe complex dysregulation of immune response in COVID-19 patients have welldocumented(32, 33). Interestingly, cells infected by SARS-CoV-2 have a lower antiviral transcriptional response marked by reduced levels of IFN-I and IFN-III levels and elevated chemokine expression compared to other respiratory virus infection, which could help to explain the delayed viral clearance despite the strong pro-inflammatory state associated with COVID-19

(34).The reduced levels of IFN-I and IFN-III could be due to short-lived nature of these cytokines in vivo(32, 34). COVID-19 patients can developlymphocytopenia, which increases the susceptibility to severe disease and to secondary infections such as pneumonia and sepsis(35).

Severe lung pathology in COVID-19 patients is associated with increased neutrophil infiltration of the lungs,as demonstratedby autopsyexamination(5, 7).Activated neutrophils release neutrophil extracellular traps (NETs)(5, 7), which contain cell-free DNA decorated with citrullinated histone 3 (citH3), myeloperoxidase (MPO), and other DAMPs such as HMGB1(36).

The presence of these molecules in NETs promotes inflammation and lung damage as demonstrated in a recent study showing increased levels of these NET components in the sera of

COVID-19 patients(7).

B lymphocytes elicit pathogen-specific adaptive immunity by producing antibodies (Abs). Two majorsubtypes of B lymphocytes have been identified: 1) B-1 cells, whichcomprise a smaller portion of total B cells,and 2) conventional B-2 cells, which comprise themajority of the B cell population.B-1 cells spontaneously produce natural IgM and, different from B-2 cells, B-1 cells do not undergo antigen recombination nor develop antigenic memory(10). Therefore, B-1 cells are not part of the adaptive immune system. In mice, the B-1 cell surface phenotype is B220dim, surface IgM (sIgM)bright, sIgDdim, CD23dim/−, CD19bright and CD43+(37). Depending on the expression of CD5 – a receptor also expressed by T cells –B-1 cells arefurther classified into either CD5+ (B-1a) or CD5− (B-1b)(10, 37).B-1a and B-1b cells originatefrom distinct progenitor cells. B-1a cells play mainly innate-like functions, serving initial defense against infectionby secreting natural Abs that protect host against acute infection or lower bacterial load; whereas,

B-1b cells secrete induced antibody needed to clear certain bacteria(10, 38, 39). For example, B-

1b cells mount acquired antibody responses to pneumonia polysaccharide-3 (PPS-3)(40).B-2 cells, on the other hand, play adaptive immune function by recognizing soluble antigens via the

B-cell receptor (BCR), undergo V(D)J recombination, class switch, and differentiate into plasma cells, which secrete ample amounts of immunoglobulin(41).

B-1a cells can neutralizea broad range of pathogens through their spontaneousand immediate release of natural IgM, while the B-1b cells or B-2 cells mount the adaptive immune response specific for each pathogen over the following weeks after infection(10, 41). The immunity produced byB-1b and B-2 cells participates in the resolution phase of the infection and leads to the formation of memory B cells, with are particularly useful in vaccine responses(10).

Copyright © 2020 by the Shock Society. Unauthorized reproduction of this article is prohibited. B-1a cells, on the other hand, are long-lived, self-renewing, and resistant to apoptosis(10). These extraordinary featuresrenderB-1a cells excellent sources ofsustainable protective immunity.

Most studies of B-1a cells have been conducted in the mouse where these cells were originally described. More recently, based on spontaneous secretion of natural IgM, constitutive activation of intracellular signaling, and efficient T-cell stimulation, human B-1a cells have been characterized as CD20+CD27+CD43+CD69 dim/−CD70dim/−(16).

B-1a cell-secretedimmunomodulatory molecules

B-1a cells are predominantly localized in serosal spaces such as the peritoneal and pleural cavities, where they constitute the majority of the total B-cells. B-1a cells are also found in spleen, bone marrow, and are hardly detectable in the blood and lymph nodes(42, 43). B-1a cells spontaneouslysecrete natural IgM, accounting for a major portion of the immunoglobulin levels in healthy individuals(44). Natural Abs form a first Ab line of defense against infection, providing protection during the period of time required for germinal center formation and production of adaptive Abs(44, 45). The natural Abs produced by B-1a cells differ from B-2 cell adaptive Abs in that they display little or no somatic hypermutation and minimal N-region addition, thus preserving theirgermline sequences(45, 46). Murine B-1a cell natural IgM is characteristically repertoire skewed, low affinity, and polyreactive(46). B-1a cell-derived natural

IgM are able to recognize phosphorylcholine (PC), an invariant constituent of the cell wall of

Gram-positive bacteria such as S. pneumoniae, as well as membranes of other bacterial pathogens, apoptotic cells, and oxidized low-density lipoprotein(47). In addition, natural Abs produced by B-1a cells recognize phosphatidylcholine (PtC), a key surface component of apoptotic red blood cells(48). Interaction of natural IgM with an infectious agent can act either

Ab-dependent cell-mediated cytotoxicity(44). Natural Abs from B-1a cells are often autoreactivewith low affinity but able to recognize surface molecules of dead and dying cellsandthus help eliminate dead cells and their debris(44). In so doing, potentially deleterious molecules are removed before setting off an uncontrolled immune cell activation that could cause tissue injury.

Apartfrom natural IgM, B-1a cells also spontaneously secrete IL-10 and, after stimulation with lipopolysaccharides (LPS), GM-CSF(13, 15). While IL-10 plays a protective anti- inflammatory role, controlling hyper-inflammation, GM-CSF promotes B-1a cells to induce IgM and IL-10 production(15, 49). In addition, B-1a cells are efficient antigen presenting cells, providing effective signaling to T-cells via MHC class II and co-stimulatory molecules

CD80/CD86, which are constitutively expressed on B-1a cells(44, 47). Furthermore, B-1a cells are able to induce T-cell proliferation and help induce CD4 T-cell differentiation.

B-1a cells regulation of neutrophil and macrophage response inARDS

Recently, the beneficial role of B-1a cells in ALI/ARDS has been demonstrated in mice with sepsis induced by cecal ligation and puncture (CLP)(50). The B-1a cell numbers were dramatically decreased in various compartments in CLP(13). Due to the reduced B-1a cells, CLP mice suffer develop hyper-inflammation and lung injury characterized by increased neutrophil infiltration, MPO production, and cellular apoptosis(13, 50). B-1a cell deficient CD19-/- mice develop particularly severe ALI after CLP(50). On the other hand, adoptive transfer with B-1a cells from syngeneic mice significantly attenuates CLP-induced ALI(50). B-1a cell release of IL-

10 decreased the production of pro-inflammatory cytokines and chemokines by the macrophages.

Copyright © 2020 by the Shock Society. Unauthorized reproduction of this article is prohibited. A key step for neutrophil migration into tissues is mediated through chemokine IL-8 and its receptor. Therefore, the reduction in chemokines mediated by B-1a cells limits neutrophil influx into the lungs and subsequent activation, as demonstrated by reduced MPO levels in the lungs of

CLP mice adoptively transferred with B-1a cells(50). MPO levels reflect not only PMN numbers, but also NET formation, because NETs contain MPO. In addition, neutrophils form

NETs in response to IL-8 and IL-1β, both of which are decreased in the lungs of CLP mice treated with B-1a cells(50). Therefore, it is reasonable to speculate that B-1a cell treatment may directly attenuate macrophage activation and NET formation in the lungs, and thus protect against ARDS.

B-1a cells and COVID-19

Immune cell profiling using single-cell RNA sequencing of COVID-19 patients demonstrated that the percentage of B cells expressing CD19, CD20, IGHD, IGHM, IL4R, and TCL1A resemblingsome of the markers of human B-1 cells were decreased (51). The reduction in B-1 cells correlated with decreased levels of serum IgM and IgD(51).Moreover, a recent study has identified potent neutralizing antibodies against SARS-CoV-2 by single-cell sequencing of B cells from convalescent patients (52). These studies suggest a deficit of B-1a cells in patients with COVID-19. B-1a cell secreted molecules participate in the innate immune response of a wide range of acute and chronic inflammatory diseases(10, 44).Below, we describe the effects ofB-1a cells in in viral and bacterial infections and outline the potential for using B-1a cells to treat patients with COVID-19.

Infection by influenza virus causes respiratory diseases in human. Mutations in the influenza virus genome generate highly variable epitopes of its surface protein, thereby making it difficult to make broadly effective vaccines against influenza virus(53). The polyreactive nature of B-1a cell-secreted natural IgM enables them to effectively eliminate the influenza virus infection in mice(54, 55). Systemic natural IgM secreted by B-1a cells is transported to mucosal surfaces through the immunoglobulin receptor conferringpartial protection against influenza virus infection(54, 55). The importance of circulatory natural IgM for protection against influenza virus has also been demonstrated in mice deficient for secreted IgMbut normal for cell surface

IgM (sIgM), which exhibited reduced viral clearance and increased mortality following influenza virus infection(55).

Surface expression of the CD5 receptor in B-1a cells plays an important role in regulating antigen-induced BCR-mediated downstream signaling(44). During influenza virus infection in mice, B-1a cell (CD5+) active responses are not dependent on antigen-induced BCR-mediated proliferation or differentiation into plasma cells, but are mainly generated by their enrichment at the lungs and adjacent lymph nodes as a result of their translocation from the pleural cavities(43,

56).Indeed, B-1a cell-mediated protection against influenza virus in mice was mediated through the activation of B-1a cells in the pleural cavity and adjacent lymph node drains, serving as a major source of local virus-neutralizing natural IgM (56). Pattern recognition receptors (PRRs) of B-1a cells recognize viral antigens to help promote their activation and translocation from the serosal cavity. B-1a cells in the pleural cavities spontaneously secrete natural Abs and other immunomodulatory molecules to confer protection against influenza virus infection(43, 56).

Copyright © 2020 by the Shock Society. Unauthorized reproduction of this article is prohibited. B-1a cells produce large amounts of natural IgM,both spontaneously and following stimulation. In mice subjected to endotoxemia, for example, the direct role of B-1 cells to attenuate the inflammatory response has been demonstrated by utilizing B-1 cell deficient Xid mice, which showed increased mortality than wild-typemice(57). In mice with CLP/sepsis, the protective role of B-1a cells is mediated through natural IgM,which neutralizes endotoxin(57,

58). Sepsis is also associated with accumulation of apoptotic cells in organs(59, 60); therefore it has been proposed that B-1a cell natural IgM may also ameliorate sepsis by facilitating the clearance of apoptotic cells by phagocytes. To evaluate the role of natural IgM in the immediate response against sepsis,a study was conducted in mice deficient in secreted IgMsubjected to acute peritonitis by CLP(61). This study revealed a significant increase in mortality in secreted

IgM-deficient mice compared with WT mice(61). This increased susceptibility was associated with increasedbacterial load and elevated levels of pro-inflammatory cytokines in the circulation.

The beneficial effect of IgM in CLP/sepsis was further demonstrated restoring resistance to CLP in secreted IgM-deficient mice reconstituted with polyclonal IgM from WT mouse (61).

COVID-19,like sepsis, is associated with extensive cell death(31),and the clearance of dying cells by natural IgM could be beneficial. Furthermore, considering the pathophysiology and symptom similarities between COVID-19 and influenza virus infection, and the B-1a cell beneficial effects in mouse models of influenza virus, it is proposed that treatment with B-1a cellswill ameliorate COVID-19.

IL-10suppressionof the cytokine storm

B-1a cells produce the anti-inflammatory cytokine IL-10 both spontaneously and after stimulation with PAMPs(10).We discussed above that the B-1a cell deficient Xid mice have poor

Copyright © 2020 by the Shock Society. Unauthorized reproduction of this article is prohibited. outcomes in CLP/sepsis. Increased levels of pro-inflammatory cytokines, TNF-α, IL-6, and decreased levels of IL-10 were found in plasma, lung, and gut inXidsepticmice(57). By utilizing macrophage and B-1a cell co-culture studies,we previously demonstrated that IL-10 production explains much of the immunomodulatory function of B-1a cells in sepsis (13). We have shown that B-1a cell frequency and numbers become decreased in CLP/sepsis, and that adoptive transfer of B-1a cells attenuates systemic inflammation, increases bacterial clearance, and improves overall survival(13). Moreover, CD19-/- mice,which have a contracted B-1a cell pool,develop sepsis of increased severitycaused by the overwhelming production of pro- inflammatory cytokines(13, 50). We have alsoshown that, in CLP/sepsis,IL-10 produced by B-1a cellscontrols the exaggerated release of pro-inflammatory cytokines in the circulation and provides overall protection. B-1a cells produce increased levels of IL-10 through the activation of the transcription factor cyclic AMP-responsive element binding protein (CREB)(13). During

SARS-CoV-2 infection, patients experience a similar cytokine storm, suggesting that it could also be amenable to control by B-1a cell-derived IL-10.

GM-CSF-mediated immune response

Following interaction with PAMPs and DAMPs with PRRs, serosal B-1a cells migrate to the spleen and lung, and differentiate to innate response activator (IRA)B cells that then contribute to the host response by producing GM-CSF(15, 49). IRABcells originate from B-1a cells; therefore, like B-1a cells, these cells are also capable of producing natural IgM when stimulated with LPS. Specific deletion of IRAB cell activity in mice impairs bacterial clearance, unleashes the cytokine storm, and precipitates septic shock(15). By contrast, treatment of septic mice with

GM-CSF producing B-1a cells attenuates inflammation, reduces bacterial load and improves

Copyright © 2020 by the Shock Society. Unauthorized reproduction of this article is prohibited. survival(15). GM-CSF is a pleiotropic cytokine that influences the production, maturation, function, and survival of monocytes and granulocytes(62, 63). GM-CSF’s role during COVID-

19remains elusive. Although GM-CSF is elevated in COVID-19 patients(6, 31), due to its rapid turnover and clearance its supplementation can be beneficial. The GM-CSF produced by the

IRAB cells inducesthe production of natural IgM in an autocrine or paracrine manner(49). As such, B-1a cells become GM-CSF producing IRA B cells, and thus have the potential to further amplify the B-1a antiviral reaction to SARS-CoV-2.

IL-35-mediated immune regulation

IL-35 is amember of the IL-12 family of cytokines which has been proposed as a potential therapeutic target in acute inflammatory and infectious diseases(64). The immunoregulatory function of IL-35 is mediated through the aid of regulatory B- and T-cells(65). A recent study showed that IL-35 caused regulatory B (Breg) cells to release both IL-10 and IL-35(66).

Injection of recombinant IL-35 or IL-35+Breg cells into mice after induction of experimental autoimmune uveitis, a mouse model of autoimmune eye disease, resolved inflammation by suppressing effector Th1 and Th17 cell responses and inducing Treg cells(66). Furthermore, IL-

35 expressing B-cellshave been shown to suppress liver inflammation associated with acute hepatitis B virus infection by regulating Th17 cell function in both humans and mice(67).

COVID-19 has also been associated with Th17 responses (68) and aberrant influx of lymphocytesto the lungs(29).Thus, the anti-inflammatory properties of IL-35 might be pivotal for developing new therapeutic approaches using IL-35+Bregs or B-1a cells for COVID-19 treatment.

T-cells are crucial to the host adaptive immune response to infection. Both exaggerated activation and anergy of T-cells are deleterious,because of promoting hyper-inflammation and launching insufficient immune response to infection, respectively. Although B-1a cells elicit immune response and natural IgM production in a T-cell independent manner, B-1a cells also play an important role in maintainingT-cell functionscommonly impaired during viral and bacterial infections(10). Conversely,IL-10 produced by B-1a cells also regulates T-cell hyperactivation. Importantly, IL-10 producing B-1 cells from the peritoneal cavity significantly reduce the severity of autoimmune colitis in mice by regulating neutrophil infiltration, naïve

CD4+ Tcell differentiation, and pro-inflammatory cytokine production duringintestinal inflammation(69). IL-35 producing regulatory B-cells regulate inflammatory T-cell responses, since IL-35 B-cells express less co-stimulatory molecules and are thus less potent at antigen- presention and T-cell activation(10, 66). Taken together, the T-cell modulatory activities of B-1a cells have the potential to reduce the exaggerated inflammation seen in the lungs and other organs of patients with COVID-19.

B-1a cell mal-function in aged animals and COVID-19’s increased severity inthe elderly

B-1a cells originate in the fetal liver and are later maintained at the serosal cavities by self- renewal with an age-related decline which has been recently implicated in the increasedsusceptibility to pneumonia of aged mice(70). B-1a cells provide immediate and sufficientprotection of mice from pneumoniathrough the production of natural IgM (10, 70). In experiments with severe combined immunodeficient mice infected with S. pneumoniae, it has been shown that the passive transfer of IgG-depleted serum from aged mice had no effect

Copyright © 2020 by the Shock Society. Unauthorized reproduction of this article is prohibited. whereas IgG-depleted serum from young mice was protective(70). The age-related decline in protective natural IgM may help to explain the disparity in COVID-19’s morbidity and mortality between young and older people(24).

B-1-related cells and their subsets and COVID-19

Various subsets of B-1a cells have been described based on phenotypic features such as program death ligand-2 (PD-L2), CD73 and PC-1(10). In addition, several other B cell subsets manifest activities that overlap with B-1a cells, including B10 cellsor Breg cells(69).

B10-cells in mice and humans are functionally defined and enumerated by their ability to express ample amount of IL-10 following ex vivo stimulation with phorbol 12-myristate 13- acetate (PMA)or LPS (71). B10 cells have also been shown to produce the anti-inflammatory cytokine transforming growth factor β (TGFβ)(14). The majority of resting peritoneal B-1a cells express PD-L2, a PD-1 ligand which is not expressed on B-2 cells(72). Although PD-L2+ and

PD-L2−B-1a cells have similar developmental and proliferative properties and autonomous immunoglobulin secretion, PD-L2+ B-1a cells express predominantlythe VH11 and VH12 genes, representing the majority of PtC-binding B-1 cells expressinggermline-likenatural IgM(72).

Asubset of B-1a cells expressing the ectoenzymesCD73 and CD39,which catalyze the extracellular adenine to adenosine, has also been shown to inhibit inflammation in mice(73).In mice, the two potent functions of B-1a cells,the innate immune andimmunoregulatoryfunctions, also differ according to their surface expressionof plasma cell alloantigen 1 (PC1), anenzyme that hydrolyzes extracellular ATP(73). PC1loand PC1hiB-1a cells express distinct immunoglobulin H repertoires and contribute differently to serum natural Abs, IL-10, and possibly other molecules relevant to B-1a cell function (73). Adoptively transferred PC1lo cells

Copyright © 2020 by the Shock Society. Unauthorized reproduction of this article is prohibited. secrete significantly higher amount of circulating natural IgM, while the PC1hi cells produce more IL-10 compared to their counterpart alloantigen expressing B-1a cells.Thus, IL-10 producing PC1hi cells stimulated with LPS or PMA negatively regulate Th1 cell differentiation.

Conversely, PC1lo cells produce antigen-specific IgM responses to pneumococcal polysaccharide antigens, while PC1hi cells do not. Thus, the PC1lo B cells are more likely to combat pathogen initially. PC1lo cells are developed from an early period of B-1a progenitors in fetal life, whereas

PC1hi cells are generated after birth(73).

Siglec-Gis a receptor expressed in mouse B-1a cells that negatively regulates the BCR signal transduction upon antigen ligation decreasingB-1a cell proliferation and IgM production(74). Siglec-G-/- micegeneratemore B-1a cells and IgM(75), but the function of these

B-1a cells and IgM have not yet been characterized. Siglec-G also negatively regulates TLR4 signaling and reduces PAMP and DAMP mediated inflammation(76). Elucidation of the function of each of these subsets of B-1a cells in patients with COVID-19 will be helpful to establish B-

1a-based treatments for COVID-19 patients.

Conclusions, limitations, and perspectives

Convalescent plasma therapy has been very promising in the treatment of COVID-19 patients(9,

31). The beneficial role of natural IgM and of B-1a cell mediators, however, have not yet been studied in the context of infection by the SARS-CoV-2. We strongly recommend that studies beconducted to assess the natural IgM contents in COVID-19 patients and determine the natural

IgM’s ability to neutralize SARS-CoV-2. In some cases, B-1a cell-mediated immune regulation might not be beneficial. B-1 cell deficient mice, for example, have shown better protection from a virulent strain of F. tularensisthan WT animals (77). It is speculated that the poorer

Copyright © 2020 by the Shock Society. Unauthorized reproduction of this article is prohibited. performance by normal mice could be the result of B-1a cell-generated IL-10 inducing immunosuppression which would limit the clearance of certain bacteria. Therefore, a finely tuned balance between B-1a mechanisms promoting immune defense and immune suppression is probably required for efficient viral clearance and recovery from infection.After more than 30 years of discovery of B-1a cells in the mouse, the surface phenotypes of human B-1a cells and

IRA B cells have been identified based on the functional aspects of murine B-1a cells(15, 16).

Controversies and disputes about human B-1a cell phenotypes, however, have limited studies of

B-1a cells in humans. As such, there is a profound deficiency of clinical data on B-1a cells in human health and diseases. With failure of over 30 clinical trials, sepsis remains a medical challenge.Learning from the failure of sepsis clinical trials, implementation of B-1a cells as a novel therapeutic tool in COVID-19 may not be easy as the pathophysiology of COVID-19 mimics with the pathophysiology of sepsis. Since natural IgM can neutralize a broad range of host molecules such as phosphatidylserine, PtC(45),studies should also focus onwhether natural

IgM can also bind to lipids in the SARS-CoV-2 membrane,as well as neutralize the S protein and prevent its binding to the ACE2 receptor. Moreover, studies should be conducted to reveal whether other molecules that are highly expressed in lung epithelial and endothelial cells like the adhesion molecules ICAM-1, PECAM-1, VECAM and integrins have any role in SARS-CoV-

2’sbindingto and penetration ofhost cells, and determine if B-1a cell therapy can inhibit SARS-

CoV-2 entry into cells by neutralizing viral binding to these molecules. When cells burst open to release newly formed virions, DAMPs are also released activating immune cells and skewingthe immune response from anti- towards pro-inflammatory(28, 29).We previously revealed eCIRPas a DAMP that induces NET-forming ICAM-1+ PMN in CLP/sepsis(78). It would be interesting to identifywhether B-1a cells opposethe effects of DAMPs on immune cell induction and

targeting DAMPs could bea rational therapeutic approach to restore B-1a cell function in

COVID-19 patients. In conclusion, these evolving new perspectives suggest a role forB-1a cells

in the pathobiology of infection by the SARS-Cov-2 virus and thetargeting of these cells as a

potentially beneficial novel therapeutic strategy to treat patients with COVID-19. The successful

implementation of treatments based on B-1a cell function may ultimately prove beneficial and

save lives.

Grants

This study was supported by the National Institutes of Health (NIH) grants R35GM118337

(P.W.) and R01GM129633 (M.A.).

Author contributions

MA originated the perspectives, gathered supportive literatures, and wrote the manuscript. MB

and PW collected literature resources, critically reviewed, and edited the paper. PW supervised

the work.

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Copyright © 2020 by the Shock Society. Unauthorized reproduction of this article is prohibited. Table-1: Comparison between pathophysiological aspects of bacterial sepsis in humans, severe COVID-19, and B-1a cell studies in CLP/sepsis.

Pathophysiological aspects Bacterial sepsis Severe COVID-19 B-1a cells in CLP/sepsis Stimuli Bacteria SARS-CoV2 Bacteria, virus Hyperimmune response Early stage At the onset (6) Release TNF-α, IL-3, (hyperdynamic induce Th17 cells (10) phase) (59) Immunosuppression Late stage Early or late stage Release IL-10, TGFβ (hypodynamic (17, 79) which can induce phase) (59) immunosuppression; regulates T cell and macrophage functions (10) Lymphocytosis/lymphocytopenia Occurs at different At the onset and after Decreased in stages of sepsis (59, recovery (51) CLP/sepsis (13) 80) Neutrophilia/infiltration in lungs Yes (59) Yes (5, 7) Controls neutrophil infiltration in lungs (50) Cytokine storm/hyperinflammation Yes (59) Yes (30) Regulates pro- inflammatory cytokines through IL-10, GM- CSF, natural IgM (10) NET formation Yes (in blood and Yes (in lungs) (5) Inhibits production of lungs) (81) MPO (a component of NETs) (50) Apoptosis Yes (T cells, B cells, Yes (T cells, B cells) Increase phagocytosis endothelial cells) (51, 52) of apoptotic cells (10) (82) Acute lung injury (ALI)/Acute Yes (83) Yes (84) Attenuates CLP/sepsis- respiratory distress syndrome induced ALI (50) (ARDS) IgM/convalescent plasma therapy IgM therapy Convalescent plasma Transfer of IgM in improves the therapy improves the secretory IgM deficient outcomes in mice, outcomes (8), while a mice or B-1a cell but in human, IgM randomized clinical deficient mice improves therapy is refractory trial shows that CLP/sepsis (61) (85) convalescent plasma does not improve the outcomes (86) Therapeutic approaches and Supportive, Supportive, antiviral Adoptive transfer of experimental interventions antibiotics, anti- drugs like Ribavirin, B-1a cells at the time of inflammatory Remdesivir, and CLP/sepsis induction cytokines, IL-7, IL- Favipiravir. was beneficial* (13) 15, anti-PD-1, Chloroquine, steroids, anticytokine Hydroxichloroquine, or immunolodulatory corticosteroids, drugs, adjunctive anticytokine or and immunoglobulin immunolodulatory therapies (87, 88) drugs (8) *B-1a cells and IRA B cells have only recently been identified in humans, and have not yet been studies in neither sepsis nor COVID-19.

Copyright © 2020 by the Shock Society. Unauthorized reproduction of this article is prohibited. Table 2: Comparison between clinical aspects of bacterial sepsis and COVID-19 and their probable response to B-1a cell therapy.

Clinical aspects Sepsis COVID-19 Probable response to B-1a therapy Contagious transmission - + Anosmia - + Cough/odynophagia +/- + Hyperthermia +/- + Myalgia/fatigue + + Diarrhea/anorexia +/- + Cytokine storm* + (robust) + At the onset of cytokine storm, B-1a cell (17) (moderate) therapy could be beneficial for (17) controlling cytokine storm (13) IL-6 release + + (higher (moderate + levels) (17) levels) (17) Immunesuppression + + B-1a cells produce IL-10, therefore at (moderate (severe the onset of immune suppressive phase lymphopenia) lymphopenia) B-1a cell therapy may be detrimental (17) (17) (10) Multiple organ dysfunction* + + + Respiratory failure/ARDS + + + Acute kidney injury + +/- ? Liver failure + + ? Cardiovascular diseases + +/- protects from atherosclerosis (89) Encephalopathy + + ? Myopathy/rhabdomyolysis + +/- ? Coagulopathy/DIC** + + ? Myocarditis/myocardiopathy* + + ? Secondary infections* + +/- protects against pneumonia (70) *conditions associated with severe COVID-19. **DIC, disseminated intravascular coagulation. ? not studied