Re-thinking the functions of IgA⁺ plasma cells

Abstract

The intestinal mucosa harbors the largest population of antibody (Ab)-secreting plasma cells (PC) in the human body, producing daily several grams of immunoglobulin A (IgA). IgA has many functions, serving as a first-line barrier that protects the mucosal epithelium from pathogens, toxins and food antigens (Ag), shaping the intestinal microbiota, and regulating host–commensal homeostasis. Signals induced by commensal colonization are central for regulating IgA induction, maintenance, positioning and function and the number of IgA⁺ PC is dramatically reduced in neonates and germ-free (GF) animals. Recent evidence demonstrates that the innate immune effector molecules tumor necrosis factor α (TNFα) and inducible nitric oxide synthase (iNOS) are required for IgA⁺ PC homeostasis during the steady state and infection. Moreover, new functions ascribed to PC independent of Ab secretion continue to emerge, suggesting that PC, including IgA⁺ PC, should be re-examined in the context of inflammation and infection. Here, we outline mechanisms of IgA⁺ PC generation and survival, reviewing their functions in health and disease.

Abbreviations

AID

activation-induced deaminase

Ab

antibody

Ag

antigen

APC

antigen-presenting cell

APRIL

a proliferation-inducing ligand

Arg

arginase

Atg

autophagy-related gene

BAFF

B-cell activating factor

bcl

B-cell lymphoma

BCMA

B-cell maturation antigen

Blimp

B-lymphocyte-induced maturation protein

BM

bone marrow

CCL

CC chemokine ligand

CCR

CC chemokine receptor

CD

cluster of differentiation

CSR

class-switch recombination

CXCL

CXC chemokine ligand

cGMP

cyclic guanosine monophosphate

DC

dendritic cell

ER

endoplasmic reticulum

FcαR

Fc fragment of IgA receptor

FDC

follicular dendritic cells

GF

germ-free

GC

germinal center

GRP

glucose-regulated proteins

GM-CSF

granulocyte-macrophage colony-stimulating factor

GALT

gut-associated lymphoid tissues

HIV

human immunodeficiency virus

IgA

immunoglobulin A

iNOS

inducible nitric oxide synthase

Id

inhibitor of DNA binding

ILC

innate lymphoid cells

IRE

inositol-requiring enzyme

IFN

interferon

IRF

interferon regulatory factor

IL

interleukin

IEC

intestinal epithelial cells

ILF

isolated lymphoid follicles

LP

lamina propria

L-Arg

L-Arginine

L-Cit

L-citrulline

L-Glu

L-Glutamate

L-Orn

L-Ornithine

L-Pro

L-Proline

LIGHT

homologous to lymphotoxin, exhibits inducible expression, and competes with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes

LT

lymphotoxinLTβR, LTβ-receptor

Ly

lymphocyte antigen

LTi

lymphoid tissue-inducer

LTo

lymphoid tissue organizing

MHC

major histocompatibility complex

MLN

mesenteric lymph nodes

NO

nitric oxide

Pax

paired box

PP

Peyer's patch

PC

plasma cells

pIgA

polymeric IgA

pIgR

polymeric Ig receptor

ROR

Retionic acid receptor (RAR)- or retinoid-related orphan receptor

IgA

SIgAsecretory

IgAD

selective IgA deficiency

SHM

somatic hypermutation

SIGNR

specific intercellular adhesion molecule-3-grabbing non-integrin-related

SC

stromal cells

TD

T-dependent

T_FH

T-follicular helper cells

TGFβR

transforming growth factor β receptor

Th

T helper cell

TI

T-independent

TLR

Toll-like receptor

TACI

transmembrane activator and calcium-modulator and cyclophilin ligand interactor

Treg

T-regulatory cell

TNFα

tumor necrosis factor α

TNFR

TNF receptor

UPR

unfolded protein response

XBP

X-box binding protein

The gut mucosa harbors the largest population of IgA⁺ PC producing large quantities of IgA that exert first-line barrier protection of the mucosa, consequently regulating the composition of the gut microbiota as well as intestinal homeostasis. We recently described a subset of IgA⁺ PC in the gut defined as IgA⁺B220^lowCD11b^lowCD11c^low Ly6C⁺Ly6G⁺ that express either TNFα and/or iNOS.¹ These cells were clearly shown to have undergone AID activation, and their presence in the gut lamina propria (LP) was dependent on the microbiota.¹ Importantly, B lineage-specific expression of the innate immune effector molecules TNFα and iNOS was shown to be required for IgA⁺ PC homeostasis at steady-state and during infection. In addition to our study, new and unexpected functions have been ascribed to IgA⁺ PC independent of Ab secretion,^2-4 suggesting that this important mucosal cell type should be re-examined in the context of inflammation and infection. We outline here mechanisms of IgA⁺ PC generation and survival, reviewing their functions in health and disease, and discuss candidate roles of iNOS and TNFα in the context of IgA⁺ PC.

Locations of IgA⁺ Plasma Cell Generation

Approximately 80% of all human PC are located in organized gut-associated lymphoid tissues (GALT) where they produce more IgA (∼50 mg/kg/day) than all other Ig isotypes combined,⁵ demonstrating that mammals devote enormous energy for continuous secretion of IgA. IgA⁺ PC release soluble IgA into internal fluids and external secretions. IgA in plasma and cerebrospinal fluids is present at lower concentrations than other Ig and is predominantly monomeric. In contrast, the composition of IgA in external secretions is heterogeneous and includes a small portion of monomers, although the majority of IgA in external secretions is polymeric IgA (dimers and tetramers - pIgA). pIgA is generated through covalent linkage by the joining (J) chain,⁶ with pIgA production first initiated by IgA⁺ PC in the mucosal subepithelium⁵ and thereafter selectively transported into external secretions⁷. pIgA binds the polymeric Ig receptor (pIgR) at the basolateral side of intestinal epithelial cells (IEC), and the complex is shuttled to the apical membrane where pIgR is cleaved to release secretory IgA (SIgA) as a hybrid molecule comprising pIgA and the secretory component provided by the pIgR.^7-9 Although mucosae are the primary inductive sites for IgA⁺ PC, in adults, about 80% of serum PC and 40% of bone marrow (BM) PC are IgA⁺, suggesting a substantial contribution of IgA⁺ PC to the long-lived PC reservoir.¹⁰

Development of intestinal IgA depends largely on commensal colonization as GF mice have much lower numbers of IgA⁺ PC.¹¹ Although massively reduced, GF animals still produce significant levels of microbiota-independent IgA, referred to as “natural IgA,” containing poly-reactive low-affinity as well as high-affinity Ab that display no evidence of somatic hypermutation (SHM).¹² Bacterial colonization of the intestine leads to oligoclonal expansion of “natural” B cell clones and induction of T cell-independent, mostly poly-reactive IgA, referred to as “primitive IgA,” sufficient for the management of commensal bacteria through immune exclusion.¹² However, it has been suggested that the fixed germ-line encoded VDJ Ab repertoire and innate immune recognition receptors are neither sufficient to deal with the constant antigenic threat of the intestinal milieu, nor adequate to support adaptation to dynamic transposable microbial communities. Evolutionary pressure exerted by such microbes has resulted in the adoption of class-switch recombination (CSR) and SHM, key functions that result in selection of high-affinity B cell clones through the activity of the enzyme Activation-Induced Deaminase (AID).¹²

The ability to make Ig at mucosal surfaces is evolutionarily conserved - observed in mammals and birds (IgA), bony fish (IgT), and amphibians (IgX).¹³ Given the large surface area of the intestinal mucosa, it is perhaps not surprising that approximately 75% of all Ig produced by mammals is IgA, primarily in the gut but also in milk and bronchial secretions.¹⁴ IgA is mainly induced in gut-draining mesenteric lymph nodes (MLN) and organized GALT structures such as Peyer's patches (PP) and isolated lymphoid follicles (ILF). PP develop during embryogenesis and contain several B cell follicles. Their formation requires the IL-7-dependent expression of lymphotoxin (LT)-α1β2 on lymphoid tissue-inducer (LTi) cells and LTβ-receptor (LTβR) expression by lymphoid tissue organizing (LTo) stromal cells (SC). Triggering of LTβR on LTo leads to the expression of chemokines such as CXCL13 and CCL20.¹⁵ Accordingly, mice that lack LTi cells (Id2^−/− or RORγτ^−/−) or mice deficient in LTα, LTβ or LTβR do not develop PP and have almost no IgA⁺ PC.^16,17 Consistent with an important role for PP in IgA induction, animals deficient in CXCR5 and CCR6, the sole receptors for CXCL13 and CCL20, respectively, have fewer PP with lower B cell numbers and exhibit reduced IgA production.^18,19 In contrast, ILF develop postnatally upon microbial colonization of the intestine. Their formation requires RORγτ⁺ innate lymphoid cells (ILC) that possess LTi-like functions.¹⁵ ILF have only a single B cell follicle but their size and number depends on microbial colonization and dietary metabolite availability.²⁰ Similar to the formation of PP, generation of ILF depends on membrane-bound LTαβ expressed by RORγτ⁺ ILC.²¹ However, T cell-dependent IgA PC induction in the LP has been recently shown to require the production of soluble LTα rather than membrane-bound LTαβ expressed by RORγτ⁺ ILC.¹⁷

Although LT-deficient mice lack LN and PP, in general these structures are not essential for the generation of IgA as IgA⁺ PC formation can occur in ILF of the LP.²² In the absence of LN and PP, LTβR signaling on radio-resistant SC is required for the expression of CXCL13 to recruit B cells to the intestinal LP. IgA induction in LTβR^−/− mice that lack LN and PP can be restored by transplantation of LTβR sufficient gut segments. This suggests that in a PP-deficient setting, LTβR signaling on radio-resistant SC is essential for supporting the generation of IgA-producing cells.²³ In contrast, it has been suggested that in the presence of PP, LTβR signaling acts downstream of other signals. Indeed, TNFR has been shown to synergize with the LT axis for IgA production in the presence of PP.²⁴ Consistent with this, LT-deficient mice have a strongly impaired immune response to Rotavirus infection, although the anti-viral IgA response eventually emerges with time.²⁵ It is possible that in the absence of LTα₁β₂/LTβR and LTα/TNFR signaling, LIGHT, an alternative LTβR ligand, may compensate. Indeed, overexpression of LIGHT results in a hyper-IgA phenotype.²⁶ Deciphering specific roles for the LTα₁β₂/LTβR, LIGHT/LTβR and LTα/TNFR axis during T-dependent (TD) and T-independent (TI) settings remains a challenging question.

It has been shown that the various structural compartments differ in their mode of IgA induction. Specifically, IgA induction in ILF can occur in the absence of T cells, suggesting that TI mechanisms of IgA induction might predominate at these sites. On the other hand, PP and MLN can support both TD as well as TI IgA induction.^27-29 Several CD4⁺ T cell subsets have been implicated in germinal center (GC)-driven IgA responses including T-regulatory cells (Treg),³⁰ T-follicular helper cells (T_FH)³¹ and Th17 cells.^32,33 It is likely that the generation of IgA⁺ PC depends on the nature of the Ag and the location of where B cells are primed. Moreover, the precise combination of different leukocyte and mesenchymal cell types might influence the generation of IgA⁺ PC. As such, the relative contributions of different anatomical compartments, cellular contributions and molecular mechanisms driving IgA production are difficult to determine experimentally and may also depend on species-specific features.

Persistence of IgA⁺ Plasma Cells

PC accumulate in particular locations during the steady state: particularly the BM, and in the case of IgA⁺ PC, in the gut. This section will describe both cell-intrinsic and cell-extrinsic mechanisms that sustain PC survival. For the most part, these mechanisms have been described for PC irrespective of isotype, although we will speculate on specific requirements for IgA⁺ PC.

Plasma cell-intrinsic mechanisms of survival

Upon encounter with Ag in the context of GC reactions, B cells up-regulate the transcription factor Bcl6 and the enzyme AID. AID is required for secondary diversification of the B cell receptor, namely SHM and CSR. Following the process of affinity maturation, B cells differentiate to become PC. This differentiation program, which requires cytokines such as IL-21 provided by Tfh, is accompanied by the down-regulation of Bcl6 and Pax5 as well as the up-regulation of the transcription factors IRF4 and Blimp-1. In both mice and humans, prdm1, the gene encoding Blimp-1, is required not only for PC formation but also Ig secretion.³⁴ More recently, it was shown that Galectin-1 expression is controlled by Blimp-1 and is expressed only by differentiating PC to support PC survival.³⁵

The rapid genetic reprogramming in Ag-experienced PC requires a massive expansion of the endoplasmic reticulum (ER) and an increase in metabolic requirements to support the biosynthetic capacity to produce large amounts of antibodies.³⁶ This leads to severe ER stress, oxidative stress and proteasome stress.^37,38 To accommodate these significant insults on cellular fitness, PC have adopted 2 cell-intrinsic mechanisms to cope with these requirements: namely autophagy and the activation of the unfolded protein response (UPR).

Autophagy is a ubiquitous catabolic process that contributes to homeostatic maintenance in eukaryotic cells and is considered a regulator of stress responses.³⁹ In times of cell stress, proteins that are not particularly useful to the cell can be captured within a double-membrane autophagosome and subsequently this cargo is delivered to a lysosome where it is degraded. The role of autophagy in dealing with intracellular pathogens in the context of infection and immunity is well appreciated.^40,41 However, the role of autophagy in the homeostasis of PC has been more recently described.⁴² Mice that exhibit a conditional deficiency in Atg5 (an essential autophagic molecule) only in B cells exhibit an enlarged ER, increased expression of Blimp1 and greater secretion of Ig. However, this enhanced production of Ig comes at a cost: PC are consequently more susceptible to cell death in vitro. Moreover, in vivo experiments showed that although the GC program in Atg5^f/fCD19^cre mice was normal, these mice are inefficient at mounting a humoral immune response and have very few long-lived PC. Thus, autophagy is one cell-intrinsic mechanism to ensure the survival of Ab-secreting PC, balancing the high cost of Ab production with cell survival.

In addition to autophagy, the UPR pathway is a stress-induced signaling cascade emanating from the ER and driven mainly by X-box binding protein 1 (XBP-1), a transcription factor required for terminal PC differentiation.⁴³ To accommodate massive Ab production by PC, the ER, which is the site of Ab biogenesis, expands. This causes ER stress and, to prevent the accumulation of unfolded proteins that can aggregate, the UPR cascade becomes activated. Specifically, accumulation of misfolded proteins can occur under cases of ER stress conditions, such as during ER expansion, to accommodate copious Ab production in PC. One model for this process is that some molecular chaperones such as GRP78 (and GRP94) are recruited to misfolded proteins within the ER lumen, liberating GRP78 from its binding partner IRE1. This results in IRE1 oligomerization and activation, thus allowing it to excise an intron of the long isoform of XBP-1 (XBP-1μ), creating a short isoform of XBP-1 (XBP-1s).

In addition to quieting ER stress by activating the UPR, XBP-1s has the added function of propagating the PC differentiation program by regulating the expression of IRF4 and Blimp1.⁴⁴ Moreover, PC survival factors are directly induced by XBP-1s including IL-6³⁷ and iNOS,⁴⁵ and nitric oxide (NO), the byproduct of iNOS activity, has also been described to have anti-apoptotic effects in different cell subsets.^46,47 Recently, it was shown that iNOS deficiency in PC leads to a decrease in both viability and an increase in caspase 3 activation, under conditions of polyclonal B cell stimulation in vitro and in vivo.⁴⁸ A recent working model has proposed iNOS as a molecule that promotes PC survival through a pathway involving NO, cGMP and protein kinase G.⁴⁹ This is consistent with our recent data showing that IgA⁺ PC are reduced in mice with a selective deficiency of iNOS in the B lineage. This implies that at least in mice, iNOS expression in monocyte lineage cells, while important for many immunological functions including IgA production,⁵⁰ is not sufficient to support IgA⁺ PC, and B cell intrinsic iNOS expression is also required.¹ PC-intrinsic survival mechanisms are depicted in Figure 1.

Figure 1.

Schematic diagram of cell intrinsic mechanisms involved in PC survival. The ER stress response, which is triggered by excessive antibody production in PC, can lead to apoptosis. To balance antibody output with survival, PC have several survival strategies, which include autophagy and the unfolded protein response (UPR). The UPR mechanism is also able to generate a short form of XBP-1 (XBP-1s), which promotes the transcription of other genes such as iNOS and IL-6, both of which are involved in PC survival.

Plasma cell-extrinsic mechanisms of survival

PC can exist for very long times, producing Abs to pre-encountered Ags for several years.⁵¹ This is a key mechanism of immunological protection for the host, yet we know very little about (1) how PC survive in these niches, (2) whether they leave these niches and (3) if so how. The BM is a major reservoir for PC.⁵² PC migrate to the BM by up-regulating CXCR4 in order to respond to CXCL12 which is expressed in the medullary cords of LN and in the BM.⁵³ Interestingly, XBP-1 has been implicated in PC migration toward CXCL12,⁴⁴ suggesting that acquisition of CXCL12 responsiveness through the chemokine receptor CXCR4 is part of the PC differentiation program. Within the BM niche itself, particular cytokines are very important for sustaining PC survival. These include BAFF, IL-6 and APRIL. BAFF and APRIL sustain PC survival by binding to the TNF superfamily member BCMA, and deficiency in BAFF, APRIL and BCMA result in a deficit of PC.⁵⁴ In addition to these cytokines, other cell types are required to sustain the PC niche in the BM, in particular eosinophils, which secrete APRIL and IL-6.⁵⁵ Given that PC can secrete pathogenic Abs in the context of some autoimmune diseases, therapies such as TACI-Ig (Atacicept), which deplete BAFF-dependent B cells as well as APRIL/BAFF dependent PC, are being designed to curtail pathogenic PC in humans. However, caution must be exercised as Atacicept worsened Multiple Sclerosis symptoms (an unexpected result given the efficacy of B cell depletion by Rituximab in the same disease setting).^56,57

In addition to the BM, the gut LP is a rich source of PC, particularly IgA⁺ PC. Specifically, B cells primed in the PP egress through the efferent lymphatics, and following recirculation through the thoracic duct, home to the small intestinal LP due to their expression of the α4β7 homing receptor as well as the CCR9 and CCR10 chemokine receptors.⁵⁸ The intestinal homing of IgA⁺ PC after vaccination is a more fined-tune process that is imprinted by the route of Ag administration. For example, immunization by the intra-rectal route can induce a higher number of Ag-specific IgA⁺ PC in the gut than the intra-nasal immunization route.⁵⁹ Once in the LP, these PC have access to survival factors that include APRIL and BAFF secreted by DC^60,61 as well as the production of APRIL from the intestinal epithelium itself⁶². Very recently it was shown that similar to what was observed in the BM, gut eosinophils, can secrete cytokines that promote IgA CSR and IgA⁺ PC survival (APRIL, IL-6), thereby supporting an IgA⁺ PC niche in the gut.⁵⁵ Whether there are specific SC types that form networks conducive to PC survival in the gut is currently unclear.

With respect to BAFF/APRIL, we have previously shown that over-expression of BAFF leads to the accumulation of IgA⁺ PC in the gut as well as other compartments. This results in a massive increase in IgA levels in the serum, and interestingly, a portion of this serum IgA appears to be specific for commensal organisms.⁶³ Mice that over-express BAFF exhibit kidney dysfunction resulting in hematuria, proteinuria and premature death.^64,65 We observed that if BAFF-transgenic mice were crossed to an IgA-deficient background, kidney pathology was largely attenuated, suggesting that over-production of IgA may have a pathological consequence not unlike in the human disease IgA nephropathy. Furthermore, commensal-specific IgA were detected in the kidneys of BAFF-transgenic mice, and re-derivation of these mice into a GF facility eliminated IgA deposition in the kidney. Thus, in these mice, BAFF drives a kidney pathology that depends on IgA and commensal bacteria. We further showed that for patients with IgA nephropathy, the related cytokine APRIL was significantly elevated in their serum.⁶³ APRIL was subsequently implicated as a top hit in a large-scale genome wide analysis of IgA nephropathy patients.⁶⁶ Thus, inappropriate survival of IgA⁺ PC, driven by aberrantly elevated levels of BAFF or APRIL, can have pathological consequences for kidney health.

Functions of IgA⁺ PC: IgA-mediated Effects

As mentioned, IgA⁺ PC are very important for maintaining intestinal homeostasis, and also play a critical role in infection and immunity. In this section, we will focus on the function of IgA itself, as well as other unanticipated functions of IgA⁺ PC in maintaining the health of the host.

B cells located in mucosal tissues play an important role in frontline defense against airway infections such as Influenza and intestinal infections including Salmonella⁶⁷ and Rotavirus.⁶⁸ The critical role of IgA in immunity is highlighted in patients with selective IgA deficiency (IgAD), defined by low serum levels of IgA. IgA deficiency is the most common primary human immunodeficiency with an estimated prevalence of 1 per 600 individuals in populations of mostly Caucasian ancestry. Although approximately 2-thirds of individuals with IgAD are asymptomatic or present only mild clinical manifestations, approximately one-third suffer from recurrent bacterial, enteroviral or protozoal infections of the respiratory and gastrointestinal tracts. IgAD patients can also present with comorbidities such as chronic intestinal inflammatory disorders and autoimmune diseases, demonstrating the critical role of IgA in regulating immune-physiologic homeostasis of the host.^69-72 IgA-deficiency in humans is often asymptomatic because in the absence of IgA, IgM can exert a compensatory function.⁷³ However, in rabbits that have been treated from birth with anti-allotypic Ab to suppress Ab production, IgA-producing cells are the first escapees.⁷⁴ Furthermore, IgA-producing B cells can emerge from mice that have a profound B cell deficiency due to their inability to express membrane-bound IgM or IgD (μMT mice). Surprisingly, μMT mice produce low levels of gut-derived IgA in response to Salmonella infection.⁷⁵ Thus, in the absence of other Ig isotypes, there is tremendous pressure to maintain IgA-producing B cells.

Studies in rodent models further illustrated that IgA exerts key functions including ‘immune exclusion’ of Ag, regulation of Ag transport and uptake, and the ability to exert immunoregulatory properties through binding to IgA receptors. Secretory IgA acts within the intestinal lumen as a first-line barrier limiting the access of intestinal Ag (e.g. food, pathogens, self-Ag) to the submucosa and circulation. IgA entraps Ag and affects microbial virulence within cells and the mucus and prevents binding to cells and dissemination.^12,76-79 IgA has been shown to bind to several receptors including FcαRI (CD89), transferrin receptor (CD71), a receptor for the secretory component, Galectin-1 and a microfold-cell IgA receptor, all of which have distinct expression patterns on myeloid, lymphoid or non-haematopoietic cells.⁶ Interaction of the Fc region of IgA with its receptors instructs and regulates intestinal innate and adaptive immunity through regulation of cellular functions upon ligation of IgA receptors. In addition, IgA has been shown to facilitate the uptake of luminal Ag across the intestinal epithelium, enhancing Ag-presentation and priming of Ag-specific immunity.^8,9,80-82

Since the gut is a complex system with 3 main interacting components, the intestinal epithelium, the microbiota and the immune system, alterations in IgA production leads to changes in the composition of the intestinal microbiota and altered intestinal barrier function. Mucosal IgA has been shown to play an important role in containing potentially invasive commensal bacteria, suggesting that IgA exerts protective effects at the level of barrier function in the gut.²⁴ This is also the case for IgA that is ingested through breast milk, and such effects of IgA on the luminal side of the gut can have a long-term impact on intestinal homeostasis beyond infancy.⁸³ Furthermore, mice lacking B cells, IgA or the pIgR exhibit changes in the intestinal microbial ecology and increased serum titers of antibodies specific for commensal and food antigens.^84-91 Bacteria in turn adapt to IgA by altering their gene expression patterns,⁹² demonstrating the critical role for IgA in regulating host-microbial mutualism.^11,85 A decrease in IgA production leads to changes in metabolic functions including lipid malabsorption and decreased deposition of body fat. This is presumably mediated by a consequential skewing of the commensal microbial composition in the absence of IgA, thus affecting energy harvest from the diet and changes in the immunological and metabolic function of IEC.^90,91,93 The molecular features of malabsorption found in B cell-deficient mice were also found in IgA-deficient mice as well as humans with common variable immunodeficiency or HIV infection, suggesting that the homeostatic and metabolic mechanisms exerted by IgA in the human gut may be similar to those observed in mice.⁹⁰ These alterations have been shown to lead to an increased risk in the development of chronic inflammatory disorders and autoimmune disease,^93,94 thus underscoring the critical role of IgA for the regulation of intestinal and systemic immune system homeostasis.

Lastly, IgA in the gut plays a key role in defense against intestinal pathogens such as Rotavirus. Rotavirus is an intestinal virus that causes diarrhea in humans and suckling mice. IEC of the small intestine are the primary site of virus replication.⁹⁵ It has been shown that IgA is important for clearance of a primary infection and for protection from re-infection in mice.⁹⁶ In humans, IgA is a good correlate of protection against Rotavirus infection, but multiple infections can occur during the lifetime of the host.⁹⁷ Recently, 2 RV vaccines were licensed, the monovalent G1P Rotarix^TM vaccine and the bovine reassortant pentavalent RotaTeq^TM vaccine. Surprisingly the efficacy of the vaccines are lower in low-income countries in Africa^98,99 suggesting that some additional gut microenvironment and host factors could be involved that need to be elucidated. Astonishingly, IgA within genital-urinary tract secretions can also exert protective effects against HIV in mucosal secretions in HIV sero-negative individuals who are highly exposed to the virus. It is thought that the particularly long hinge region of IgA may have contributed to such neutralizing effects.¹⁰⁰ However, it is clear that there is significant affinity maturation of the IgA response in the GALT, signifying that Ag-driven selection events are the dominant force for shaping the IgA repertoire.¹⁰¹ Further study of the broad neutralizing functions of some IgA responses is nevertheless warranted as a consideration for vaccine design.

Functions of IgA⁺ PC: Beyond Antibody Production

We recently showed that the presence of IgA⁺B220^lowCD11b^lowCD11c^lowLy6C⁺ Ly6G⁺ cells that express either TNFα and/or iNOS require a microbiota for their persistence in the gut.¹ Our observations are in accordance with a recent report that describes a unique microbe-dependent subset of IgA⁺ PC, mainly characterized as CD38⁺CD138⁺ Blimp-1⁺, which express CD11b and require IL-10 for their maintenance of IgA production.¹⁰² These findings suggest that in the process of differentiating to become IgA⁺ PC, the gut environment may have endowed IgA cells with ‘monocytic’ potential, a phenomenon that has been previously observed in.vitro¹⁰³ Moreover, B cells display non-conventional monocytic functions in other species¹⁰⁴ and in response to TLR ligation.¹⁰⁵ Previously, a subset of splenic B cells that regulate T cell function through their expression of indoleamine 2,3-dioxygenase was shown to express CD11c.¹⁰⁶ As myeloid cells and B cells share a common developmental pathway,^107-109 it is possible that maintaining CD11c expression confers a selective advantage to specific B cell subsets. Although the expression of Ly6C and Ly6G are most frequently used to identify inflammatory monocytes and neutrophils, lymphocytes have been shown to express these proteins as well. Splenic and LP PC express Ly6C and its cross-linking positively regulates Ig production.¹¹⁰ In addition, Ly6C supports homing of central memory CD8⁺ T cells,¹¹¹ and Ly6G ligation coordinates the recruitment of neutrophils in vivo,¹¹² suggesting that expression of Ly6C and Ly6G by IgA⁺ PC might regulate their positioning and function. Importantly, expression of TNFα and iNOS in B lineage cells was shown to be critical for the maintenance of IgA levels as well as intestinal immune homeostasis at steady-state and after immune challenge with a gut-tropic pathogen,¹ suggesting a functional synergy of these 2 innate immune mediators for the regulation of intestinal PC functions.

The role of iNOS in PC

Inducible NOS (iNOS or NOS2) is one of 5 intracellular enzymes (neuronal NOS, endothelial NOS, arginase 1 (Arg1) and Arg2) that utilize the extracellular amino acid L-Arginine (L-Arg) to modulate immune responses.^113,114 L-Arg is a central intestinal metabolite, functioning both as a constituent of protein synthesis and as a regulatory molecule limiting intestinal alterations and maintaining immune-physiological functions.^115,116 While the cytosolic Arg1 is abundant in liver as part of the urea cycle, Arg2 is mostly abundant in extra-hepatic tissues such as the kidney, brain and gut. Arg2 is also highly expressed in haematopoietic cells and important for the production of L-Ornithine (L-Orn), L-Proline (L-Pro) and L-Glutamate (L-Glu). In contrast NOS catalyzes the production of NO and L-Cit from L-Arg.¹¹³ L-Arg is derived from the diet, turnover of proteins and endogenous production through synthesis from L-citrulline (L-Cit) and enzymes of the urea cycle. Although synthesis of L-Arg from L-Cit can occur in many cell types, a major part of endogenous synthesis occurs via "the intestinal-renal axis" involving IEC of the small intestine and proximal tubule cells of the kidney. Importantly, L-Cit is produced primarily by IEC from NH₃, CO₂, and ornithine by enzymes of the urea cycle, and is supplied to the kidney and other tissues for synthesis of L-Arg.^116-118 While in healthy adults the level of endogenous L-Arg synthesis is sufficiently great that L-Arg is not an essential amino acid, catabolic stress can lead to hypoargininemia, a condition where levels of endogenous L-Arg may not suffice to meet metabolic demands, impacting on the host's immune response.

L-Arg availability in the alimentary tract plays a key role for intestinal homeostasis during inflammation and infection.¹¹³ Infection-associated L-Arg deficiency has been shown to contribute to immunopathology and predisposes to co-infections,¹¹⁹ and clinical trials involving L-Arg administration have shown substantial decreases in inflammation and infectious complications.¹²⁰ L-Arg supplementation attenuated tissue damage in intestinal ischemia and promoted healing of the intestinal mucosa.^121,122 In addition, in a rodent model of dextran sulfate sodium-induced colitis, oral L-Arg supplementation dampened pro-inflammatory responses and inflammatory cell infiltration, improved mucosal barrier integrity and enhanced IEC migration in an iNOS-dependent manner.¹²³ In addition, infection with Citrobacter rodentium was shown to cause a significant decrease in serum L-Arg concentration with associated infection-induced immunopathology that was partially reversed after L-Arg supplementation.¹²⁴ Although, their distinct contribution remains to be explored, it is likely that altered activities of L-Arg catabolizing enzyme families, Arg as well as NOS all contribute to the observed intestinal immunopathologies. It therefore can be postulated that Arg activity through conversion of L-Arg into L-Orn enhances intestinal epithelial barrier function,¹²⁵ while iNOS activity confers an anti-inflammatory state through regulation of energy metabolism.¹²⁶

Indeed, iNOS activity dampens inflammation through regulation of myeloid and lymphoid cell activation. NO produced by iNOS in inflammatory monocytes and DC regulates cellular metabolism, inflammatory cytokine production, survival and responsiveness to chemotactic signals.^127-130 While several reports demonstrate that NO counteracts the expansion of activated CD4⁺ (Th1, Th2 as well as Th17) and CD8⁺ T cells,^131-135 endogenous iNOS activity was shown to be required for the polarization of Th17 and Treg cells,^134,136 suggesting that the magnitude and localization of iNOS activity and NO production is critical for the priming and polarization of the Ag-specific T cell pool. Moreover, iNOS-deficient animals have been shown to have lower serum and intestinal IgA and serum IgG2b.⁵⁰ iNOS-expressing DC have been suggested to influence IgA CSR through induction of BAFF and APRIL production, as well as through regulating the expression of TGFβRII in B cells.⁵⁰ However, mice that constitutively lack DC display slightly increased levels of IgA compared to controls, indicating that other cell types besides DC are essential sources of iNOS and TNFα.¹³⁷ Our recent observations demonstrate that intrinsic iNOS expression by B cells is required for IgA⁺ PC homeostasis.¹ Although our data do not eliminate the possibility that complete deletion of iNOS in all other stromal and haematopoietic cell types would have an impact on IgA production, our results show that the expression of iNOS in B-lineage cells is essential for the homeostatic production of IgA within the small intestine.¹ Our observations are in agreement with a recent report demonstrating that iNOS expression in B cells positively influences the expression of AID.¹³⁸ In addition to its central role in regulating intestinal Ab responses, iNOS expression by B cells appears to have a critical role in regulating intestinal inflammation and antibacterial defense to Citrobacter rodentium, as animals selectively deficient for iNOS in B lineage cells exert bacterial translocation to the spleen, enhanced weight loss and intestinal pathology.¹. This may explain why B-cell-deficient mice, but not IgA^−/− mice or mice that lack secreted IgM, also fail to control Citrobacter rodentium.¹³⁹

The role of TNFa

B cell-derived TNFα has been shown to regulate architecture of lymphoid organs and mucosal Ab-dependent and Ab-independent immune defense mechanisms. Secondary lymphoid organs in TNFα-deficient mice lack primary B cell follicles and FDC clusters, fail to form GC and show an altered marginal zone.¹⁴⁰ Some TNFα^−/−- as well as TNFR1^−/−-deficient mice do not develop PP, while others only develop fewer and smaller PP.^141-143 Importantly, B cell-derived TNFα is critical for the formation of FDC networks, GC and B cell follicles in the spleen, while TNFα from B and T cells synergizes for GC and FDC networks in LN, which is critical for the generation of local immune responses.^144,145 Moreover, maintenance of long-lived PC in BM is thought to require a specialized niche that mediates their survival at least partially through TNFα.¹⁴⁶ Lastly, full maturation of ILF requires TNFRI,²¹ and B cell-derived TNFα was also shown to regulate GC in PP.¹⁴⁷ Our recent work revealed a critical role for TNFα and iNOS expression by B cells for the generation of IgA⁺ PC.¹ Considering that T cell-dependent and T cell-independent IgA induction mainly occurs in GC of PP and ILF, respectively, it is possible that TNFα from plasma cells contributes towards the organization of PP and ILF architecture.

B cell-derived TNFα has been recently shown to regulate mucosal immune responses. The ability of B cells to produce TNFα depends on the stimulus used for activation and the developmental state of the cells. For example, CD40-activated B cells do not make TNFα, while B cells activated first by Ag and then with Ag and CD40L make TNFα.^148,149 Memory B cells, but not naïve B cells, secrete TNFα following dual stimulation with anti-Ig and CD40L.¹⁴⁸ Moreover, only CD27⁺ memory B cells produce and TNFα following TLR1/2 activation.¹⁵⁰ B cell-derived TNFα is required for the generation and maintenance of Heligmosomoides polygyrus-specific PC.¹⁵¹ Moreover, B cell-derived TNFα amplifies IFNγ production by T cells during Toxoplasma gondii infection,¹⁵² demonstrating a critical role of B cell-derived TNFα for the regulation of mucosal immunity.

Plasma Cells As A Source of Cytokines

There are accumulating examples of unexpected functions of Ab-secreting PC (Fig. 2). For example, an innate and rapid anti-microbial response has been shown to be associated with the production of GM-CSF by plasmablasts.⁴ In addition, a PC population has been shown to produce IL-17A in response to a parasitic infection.³ The accumulation of IgA⁺ PC in some disease settings such as athlerosclerosis¹⁵³ implores one to consider if IgA⁺ PC in these locations are exerting effects via their secretion of Abs or perhaps via other Ab-independent functions or both. In addition to their role in pathogenesis (such as in the BAFF-transgenic mouse) and in protection against pathogens (such as Rotavirus infection) it is important to consider PC, and perhaps IgA⁺ PC as putative regulatory cells. As mentioned, Atacicept, which will affect both the B cell compartment as well as the PC compartment, had an unexpected harmful effect on Multiple Sclerosis patients in contrast with Rituximab treatment, which exhibited clear clinical benefit. PC express MHC class II, and they have been shown to present Ag to T cells while inhibiting T_FH function.¹⁵⁴ In addition, it has been recently shown that PC can make IL-10 and IL-35 to attenuate neuroinflammation in an animal model of Multiple Sclerosis.² Whether such regulatory functions will be the case specifically for IgA⁺ PC derived in the gut remains unclear. However, it is important to note that IgA cannot fix complement, and IgA itself can also dampen immune responses by inducing regulatory dendritic cells by binding to the cell surface receptor SIGNR1,¹⁵⁵ suggesting that while these cells provide effective protection at mucosal barriers, they may co-provide regulatory functions to prevent excessive inflammation. Putative functions of PC and IgA⁺ PC in particular are depicted in Figure 2.

Figure 2.

Rethinking PC function. This figure depicts both general antibody-dependent and antibody-independent functions of PC as well as specific functions of IgA and IgA⁺ PC.

Conclusions

In general, we associate IgA⁺ PC with mucosal tissues. However, we know that there are IgA⁺ PC in the BM. Were these PC generated in the periphery, or within mucosal tissues? Can IgA⁺PC migrate to other sites? Certainly, recent work has suggested that the PC compartment is more dynamic than we originally supposed in terms of their egress from the BM, particularly when there is inflammation in the periphery.¹⁵⁶ Moreover, IgA⁺ PC have been found in non-mucosal tissues such as the adventia of the vascular wall.¹⁵³ Our work has implicated IgA in kidney pathogenesis, and an important question is whether IgA⁺ PC themselves are found in this location.⁶³ Moreover, although the cecum and colon are relatively poor in IgA⁺ PC in the steady-state, in responses to Citrobacter rodentium IgA⁺ PC quickly accumulate in the cecum/colon in as little as 36 hours.¹ The fact that in the steady state approximately 40% of PC in human BM are IgA⁺ PC that express CCR4 as well as the β7 integrin and CCR10 may suggest a contribution of mucosal PC to the BM compartment, however more research is needed to unambiguously determine where these PC were originally generated.¹⁰ The functional role of IgA PC, whether regulatory or pro-inflammatory, in extra-intestinal sites will be important to discern in both healthy and diseased states.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

JLG is supported by operating grants from the Canadian Institutes of Health Research, the Multiple Sclerosis (MS) Society of Canada, the MS Society Research Foundation and the Kidney Foundation. OLR is supported by a post-doctoral fellowship from the MS Society of Canada. JHF acknowledges funding by the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada.