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The Evaluation of Cytokines to Help Establish Diagnosis and Guide Treatment of Autoinflammatory and Autoimmune Diseases

Anne-Laure Chetaille Nézondet1,2, Patrice E. Poubelle1,3, Martin Pelletier3,4,5

1Département de médecine, Faculté de Médecine, Université Laval, Québec, QC, Canada. 2Axe de recherche Reproduction, santé de la mère et de l’enfant, Centre de recherche du CHU de

Québec-Université Laval, Québec, QC, Canada. 3Axe de recherche sur les maladies infectieuses et immunitaires, Centre de recherche du CHU de

Québec-Université Laval, Québec, QC, Canada. 4Département de microbiologie-infectiologie et d’immunologie, Faculté de Médecine, Université

Laval, Québec, QC, Canada. 5Centre de recherche en arthrite de l’Université Laval – ARThrite (Arthrite Recherche

Traitement), Université Laval, Québec, QC, Canada.

Correspondence Dr. Martin Pelletier

CHU de Québec-Université Laval Research Center, Room T1-49

2705 Boul. Laurier, Québec, QC, G1V 4G2, Canada.

Email: martin.pelletier@crchudequebec.ulaval.ca

Running Title: Cytokine patterns in autoinflammation and autoimmunity

Summary sentence: Review on the use of cytokines to help diagnose and guide treatments of

patients suffering from autoinflammatory and autoimmune diseases.

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Abbreviations

AIM2: absent in melanoma 2

AP-1: activator protein-1

ASC: apoptosis-associated speck-like protein containing a CARD

CAPS: cryopyrin-associated periodic syndrome

CARD: caspase activation and recruitment domain

CINCA: chronic infantile neurologic, cutaneous, articular

DMARD: disease-modifying anti-rheumatic drug

FCAS: familial cold autoinflammatory syndrome

FMF: familial Mediterranean fever

IFN: interferon

IL: interleukin

IL-1Ra: IL-1 receptor antagonist

JAK, janus kinase

LRR: leucine-rich repeat

MAPK: mitogen-activated protein kinase

MWS: Muckle-Wells syndrome

NF: nuclear factor

NLR: NOD-like receptor

NLRC: NOD-LRR-CARD-containing

NLRP: NLR-LRR and pyrin domain-containing

NOD: nucleotide-binding oligomerization domain

NOMID: neonatal-onset multisystem inflammatory disease

PBMC: peripheral blood mononuclear cell

pDC: plasmacytoid dendritic cell

PMA: phorbol myristate acetate

PsA: psoriatic arthritis

RA: rheumatoid arthritis

SLE: systemic lupus erythematosus

SNP: single nucleotide polymorphism

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STAT, signal transducer and activator of transcription

TNF: tumor necrosis factor

TNFR: TNF receptor

TRAF: TNFR associated factor

TRAF3IP2: TRAF3 interacting protein 2

TRAPS: TNFR-associated periodic syndrome

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Abstract Our knowledge of the role of cytokines in pathological conditions has increased considerably

with the emergence of molecular and genetic studies, particularly in the case of

autoinflammatory monogenic diseases. Many rare disorders, considered orphan until recently,

are directly related to abnormal gene regulation, and the treatment with biological agents

(biologics) targeting cytokine receptors, intracellular signalling or specific cytokines improve the

symptoms of an increasing number of chronic inflammatory diseases. As it is currently

impossible to systematically conduct genetic studies for all patients with autoinflammatory and

autoimmune diseases, the evaluation of cytokines can be seen as a simple, less time-consuming

and less expensive alternative. This approach could be especially useful when the diagnosis of

syndromes of diseases of unknown etiology remains problematic. The evaluation of cytokines

could also help avoid the current trial-and-error approach, which has the disadvantages of

exposing patients to ineffective drugs with possible unnecessary side effects and permanent

organ damages. In this review, we discuss the various possibilities, as well as the limitations of

evaluating the cytokine profiles of patients suffering from autoinflammatory and autoimmune

diseases, with methods such as direct detection of cytokines in the plasma/serum or following ex

vivo stimulation of peripheral blood mononuclear cells leading to the production of their

cytokine secretome. The patients’ secretome, combined with biomarkers ranging from genetic

and epigenetic analyses to immunological biomarkers, may help not only the diagnosis but also

guide the choice of biologics for more efficient and rapid treatments.

Introduction

The term "cytokine" (from Greek κύτταρο/cell, and κίνηση/movement) was proposed by Stanley

Cohen in 1974 to broaden the concept of intercellular mediators produced solely by lymphocytes

(lymphokines) or monocytes (monokines) after he showed that migration-inhibitory factor was

not only produced by lymphoid but also by non-lymphoid cells [1-3]. Cytokines represent a

broad group of peptides responsible for intercellular communications and signalling that regulate

immunological, hematological, virological and cell biological responses. Cytokines gather

interferons (IFNs), interleukins (ILs), chemokines, colony-stimulating and growth factors. The

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very first cytokine discovered back in 1957 was an “interfering agent” produced by leukocytes in

the presence of viruses and named IFN by Isaacs and Lindenmann [4]. To date, the last cytokine

discovered is IL-38 [5]. Amongst their multifaceted functions, cytokines are involved in immune

regulation, inflammation, generation of blood cells, chemotaxis, cellular growth and

differentiation. Cytokines are also pleiotropic and redundant multifunctional factors that can act

in synergy. As such, they can exert pro- or anti-inflammatory properties, or both, depending on

the cells and the tissues targeted, and the action of some cytokines is regulated by endogenous

antagonists [6]. At the cellular level, cytokines act through specific receptors that activate

intracellular signals; this scheme leads to, at least, three levels of therapeutic targets: cytokines

themselves, cytokine receptors or common receptor units, and intracellular signals.

Since the advent of molecular and genetic studies, our understanding of the implication of

cytokines in multiple diseases has dramatically increased, especially regarding monogenic

diseases and autoinflammation. Their clinical value regularly increases since their modulation by

selected antibodies to neutralize the cytokine or its receptor, as well as by molecules that target

intracellular signals, alleviates an increasing number of diseases and rare disorders considered

orphan until recently and directly related to their abnormal genetic regulation. Interestingly,

immunological diseases have been presented as a continuum of multiple disorders, from

polygenic to monogenic autoinflammatory and autoimmune diseases [7]. This classification

allows locating the part of autoinflammation and autoimmunity in diverse diseases considered

immunological diseases (Figure 1), a denomination that is too vague. Conceptually, this

continuum from polygenic to monogenic diseases allows better targeting of the treatment that

will improve the “autoinflammation” component of a specific disease. In this regard and given

the complexity of the specific immunological diseases related to each patient, the best treatment

to consider should reflect on the particular cytokine(s) implicated in the patient's disease. This

domain of therapeutic options and causal investigations is in constant expansion due to more and

more in-depth knowledge of the mechanisms associated with autoinflammation and

autoimmunity.

The present review aims at summarizing the options for evaluating the cytokine(s) implicated in

a patient’s condition, keeping in mind whether biological agents (biologics) are available.

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Moreover, one of the objectives of this review is to emphasize the role of cytokine evaluation in

helping problematic diagnoses of diseases of unknown etiology. The current definitive tests for

autoinflammation and autoimmunity are commercially-available whole-exome sequencing-based

screening panels. These clinically-certified tests are reasonably cost-effective ways to screen

hundreds of genes and provide clinical guidelines, especially for diseases with known mutations.

However, it is presently impossible to systematically conduct such genetic studies for all patients

with autoinflammatory and autoimmune diseases. Also, a vast number of patients with polygenic

autoinflammatory immune diseases will have a weak and non-contributive genetic investigation

compared to the meager number of rare monogenic disorders with a specific genetic abnormality

easily detectable. In this regard, measurement of cytokines in plasma and after leukocyte

activation can be seen as a simple, less time-consuming and less expensive alternative that merits

extensive studies for validation in clinical settings.

Cytokines implicated in autoinflammation

Historically, the concept of autoinflammation emerged from the discovery of the genetics related

to familial Mediterranean fever (FMF), an inherited disease associated with mutations in the

gene MEFV that encodes the protein Pyrin (initially termed "marenostrin" from the Latin name

of the Mediterranean sea, mare nostrum) [8, 9]. In fact, autoinflammation, a term that defines the

activation of the innate immune system without any infection but related to abnormal cytokine

overproduction, has been coined for the first time to the monogenic inherited syndromes

associated with mutations in the 55 kDa tumor necrosis factor receptor (TNFR1) and entitled

TNFR1-associated periodic syndrome (TRAPS) [10]. Since this period and due to the expansion

of genetic studies, the field of autoinflammatory diseases has exploded with a better knowledge

of rare monogenic autoinflammatory diseases as well as polygenic autoinflammatory disorders

such as inflammatory bowel diseases (Crohn’s disease and ulcerative colitis), gout, some

categories of reactive arthritis and psoriasis/psoriatic arthritis (without MHC associations) and

idiopathic uveitis [7].

One of the oldest known autoinflammatory diseases can be linked to IFN, as they have been

reported and studied for nearly a century, although the link between autoinflammation and these

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diseases was established less than 20 years ago [4, 11]. Despite their considerable heterogeneity,

they were gathered under the term interferonopathies coined in 2011 by Yanick J. Crow [12].

This group of diseases, the prototypic one being the Aicardi-Goutières syndrome, brings together

all the monogenic type I interferonopathies (IFN-α and -β) that have substantially benefited from

genetic investigations and biological assays to explain the complexity of these diseases [11, 13,

14]. In this regard, the evaluation of IFN concentrations in media from peripheral blood

mononuclear cells (PBMCs) was always disappointing due to undetectable amounts of this

cytokine. Even if all cell types can produce type I IFN, plasmacytoid dendritic cells (pDCs) are

the main cell type responsible for the production of type I IFN in the blood [15, 16]. However,

blood DCs correspond to at most 1% of mononuclear cells, which represent about 8% of total

blood leukocytes, and pDCs can reach at best 0.38% of mononuclear cells among DCs [17].

Associated with the meagre number of pDCs among PBMCs, the rapid appearance and

disappearance of type I IFN in the blood make the dosage of this cytokine almost impossible or

waiting for more validated sensitive methods of cytokine dosage. Investigations remain based on

signalling leading to interferon-stimulated genes, neutralization assays of interferon activity or

interferon bioactivity such as viral cytopathic assays. It is also useful to note that type I IFN is

greatly implicated in classic immune diseases such as systemic lupus erythematosus (SLE)

during which PBMCs from flare periods overexpress IFN-regulated genes, as studied by

oligonucleotide microarrays [18].

One of the most significant improvements in the knowledge of autoinflammation dates back to

the beginning of the 21st century with the discovery of a multimeric protein complex responsible

for the activation of caspase-1 known to cleave pro-IL-1β or pro-IL-18 into active compounds

[19]. At the same time, rapid clinical improvement associated with the treatment by IL-1 receptor

antagonist (IL-1Ra) given to patients with the Muckle-Wells Syndrome (MWS) allowed

clarifying the role of members of the IL-1 family in the pathogenesis of this disease [20]. MWS

belongs to the cryopyrin-associated periodic syndrome (CAPS), a group of three rare hereditary

autoinflammatory diseases including, besides the MWS, the familial cold autoinflammatory

syndrome (FCAS) and the neonatal-onset multisystem inflammatory disease (NOMID)/chronic

infantile neurologic, cutaneous, articular (CINCA), in which the above multimeric protein

complex presents defects leading to overproduction of IL-1β associated with inflammatory

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symptoms recorded in CAPS [21]. Interestingly, monocytes from these patients were shown to

overproduce IL-1β [22]. Since then, studies at the functional and genetic levels have focused on

this multiprotein complex, named inflammasome. These investigations have led to the grouping

of various orphan diseases with similar pathogenesis of autoinflammation, the

inflammasomopathies, in which the overproduction of cytokines, especially members of the IL-1

family, is a typical pattern. Presently, the members of the IL-1 family can be summarized as

seven agonistic actors (IL-1α and IL-1β, IL-18, IL-33, IL-36α, IL-36β, IL-36γ), three

antagonistic actors (IL-1Ra, IL-36Ra, IL-38), and one anti-inflammatory actor (IL-37). At a

glance, inflammasome components are sensor molecules NLRP1 [NOD(nucleotide-binding

oligomerization domain)-like receptor (NLR)-LRR(leucine-rich repeat) and pyrin domain-

containing 1], NLRP3, NLRP6, NLRP12, NLRC4 [NOD-LRR-CARD[caspase activation and

recruitment domain]-containing 4]), Pyrin, the adaptor molecule ASC (apoptosis-associated

speck-like protein containing a CARD) present in all inflammasomes, and the effector molecule

caspase-1 [23].

In the last two decades, the knowledge about the inflammasomes has dramatically evolved. This

has led to the differentiation of several previous orphan diseases being regrouped into new

syndromes defined by their genetic associations (see recent reviews [24-27]). These new

syndromes share an overproduction of proinflammatory members of the IL-1 family, in

particular IL-1β and IL-18, that could be present in the blood or the conditioned media from

PBMCs of patients. This new era of research is rapidly expanding due, in part, to novel

techniques such as next-generation sequencing and the extensive collaboration between

geneticists and clinicians [28]. As an example, recent reports indicated that blocking IL-18 with

an anti-IL-18 antibody failed to improve type II diabetes, even if well-tolerated, but could be

very useful in treating inflammatory bowel diseases in which IL-18 is involved through

mutations in the NLRC4 gene [29-31].

Cytokines involved in autoimmunity

In contrast to autoinflammation, in which self-directed inflammation is driven by dysregulation

of molecules and cells of the innate immune system, autoimmunity leads to self-directed

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inflammation through aberrant responses of adaptive immune cells (essentially B and T cells),

resulting in a break of tolerance and immune reactivity to native antigens. A representative

disease of autoimmunity, along with diseases such as type 1 diabetes, Graves’ disease and

myasthenia gravis, is probably SLE, considered for a long time as a common autoimmune

disease with abnormal activation of autoreactive T and B lymphocytes [32, 33]. However, SLE

can be considered as an example in which specific cytokines are responsible for an

autoinflammatory process, in particular type I IFN, despite the absence of bacterial and viral

components [18, 34]. This multifactorial and multisystem autoimmune disease affects mainly

women (9:1 female to male ratio) and is characterized by the appearance of antinuclear and anti-

DNA autoantibodies. Endosomal TLRs, in particular TLR 7 and 9 present in pDCs, cells which

are specialized in type I IFN generation, are receptors for DNA and RNA. Activation of these

TLRs on pDCs leads to increased IFN (especially IFN-α) followed by events downstream of IFN

receptors. Endpoints of this activation are autoinflammatory symptoms that affects multiple

organs in SLE [35]. To this end, evaluation of type I IFN with accurate and very sensitive

ELISAs could be of great interest. The pathogenesis of autoimmune diseases has definitively

evolved since the initial scheme of Th1- and Th2-associated diseases, especially regarding the

implication of the Th17 cells as well as the IL-12/IL-23 pathways in various autoimmune

diseases [36]. SLE pathogenesis is characterized by dysregulation of IL-2 and other members of

the IL-2 superfamily such as IL-15 and IL-21, but also dysregulation of members of the IL-12

family, in particular IL-27. Patients with SLE produce less IL-2 and IL-27 than healthy subjects.

Another finding regarding SLE is the potential role of IL-17 in its pathogenesis [37-41] (see

recent reviews by Li et al. [42] and Koga et al. [43]). Other cytokines such as IL-6, TNF and

TNFRs, BAFF/APRIL, IFN-γ, IL-18, IL-21, IL-10 have been reported elevated in sera of

patients with active SLE, whereas IL-1Ra has been found decreased [35]. This complex network

of cytokines in SLE is an excellent rationale to study the secretome of PBMCs from SLE

patients, active or not, to get an exact profile in the hope of finding new efficient therapies for

this disabling disease.

Several other autoimmune diseases affecting many persons have relatively common pathogenesis

driven by Th1 and Th17 cells, namely psoriasis, psoriatic arthritis (PsA) and rheumatoid arthritis

(RA) [44, 45]. These specific cytokine networks also exist in inflammatory bowel diseases and

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multiple sclerosis, although they are adapted to mucosal and brain inflammation, respectively

[46, 47]. The expanding number of biologics used to improve these autoimmune diseases

confirms the major role of cytokines in autoimmunity. However, the major problem remains the

choice of the right biologic for individual patients. Despite initial evidence for the roles of

cytokines in the immunopathogenesis of the above autoimmune diseases that gave prominent

value to IL-2, IFN-γ, TNF and IL-1, many years of clinical trials with large cohorts of patients

have shown that targeting TNF was effective at controlling several diseases. Even if the heavy

use of anti-TNF biological agents has revolutionized the treatment of RA, 30 to 40% of patients

do not respond to or become resistant to the treatment [48-50]. More recent clinical trials suggest

that IL-6 also represents an excellent target in RA as well as in several other immune disorders

[51-54]. It is noteworthy that, concerning the expanding knowledge of the mechanisms of these

autoimmune diseases, numerous biologics emerged as efficient treatments. This further

demonstrates the complexity of the immunopathogenesis of autoimmune diseases and the

increasing demand for a personalized therapeutic approach to avoid trial-and-error treatments. In

addition, environmental and genetic factors can modify the final expression of the immune

diseases, that are presently unpredictable. So, concerning the multiple cytokines implicated and

the expanding number of specific biologics, the knowledge of each patient's cytokine secretome

can surely provide beneficial information, which is also applicable to small molecules that target

components upstream of cytokine signalling such as janus kinase (JAK)-signal transducer and

activator of (STAT) inhibitors. Many cytokines presently known act through JAK signals, apart

from, for instance, members of the IL-1 or TNF families. JAK inhibitors (jakinibs) belong to a

new therapeutic era in expansion. Jakinibs should target JAK1, 2, 3 and TYK2 signalling

pathways that transmit signals from variable cytokines (see review [55]) and, as such, the

knowledge of each patient's cytokine secretome will help to choose the appropriate jakinib or

biologic.

To summarize the present knowledge of the cytokines involved in these autoimmune diseases

and that can be a therapeutic target, it is justified to mention TNF, members of the IL-1 family,

IL-6, GM-CSF, members of the IL-17 family, IL-23, IL-12, IL-21, IL-15, IL-22, IL-26 without

forgetting possible new members to be discovered [46, 56-58]. However, it is noteworthy that

specific subgroups of patients will respond to certain biologics while others will not. For

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example, treatments with IL-1Ra, IL-18 binding protein, or tocilizumab were reported to be

efficient in a minority of patients with inflammatory bowel diseases [58]. Similar variability is

present in patients with psoriasis, PsA and RA [56]. In conclusion, independently of the type of

autoimmune diseases, the cytokine network is too complex to hope an appropriate, but empiric,

biologic treatment. Moreover, to avoid negative results obtained from the analysis of plasma or

serum, a cell’s secretome will rapidly, and at best, give invaluable insight into the possible

abnormalities, which, combined to biomarkers (clinical, biochemical, genetic, epigenetic,

genomic, immunological) [59-63] could not only help the diagnosis but guide the choice of

biologics for more efficient and rapid treatments.

Why, when and how to evaluate cytokines?

The main question presently could be whose patients are relevant to evaluate their cytokine

secretome and when the information could be the most relevant. Acute inflammatory symptoms

such as a rash (i.e. urticaria, psoriasiform rash, pseudo-erysipelas, cellulitis), arthritis, serositis,

fever, aphthous ulcers, headache, hearing loss, edema, should lead to an investigation of the

patient’s cytokine secretome, particularly in children or young adults. Skin symptoms are often

the first clinical manifestation of autoinflammatory diseases and can include urticarial exanthema

(neutrophilic dermatosis), pseudo-urticaria, dermo-hypodermitis, psoriasiform lesions,

granulomatous dermatitis and aphtosis [64]. The rationale for the evaluation of the cytokine

secretome of these patients is at the levels of the possible identification of the factor responsible

for the inflammatory symptoms leading to the rapid choice of appropriate treatment, as in-depth

genetic investigation for rare monogenic disorders leads too often to inconstant genetic

mutations. Chronic autoimmune diseases that require a biologic treatment can also benefit from

an investigation of their cytokine secretome to target the cytokine with the most potent

therapeutic effect. The best time to investigate the secretome would probably be during a flare or

at the time of diagnosis and before treatments, as cytokine levels are often found to be increased

in these conditions [65-67]. Most of the time, patients are examined during the chronic phase of

their autoimmune disease and justify several samples to obtain longitudinal data and to hope the

most exact pattern of their cytokine secretome knowing that their cytokine pattern evolves with

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time [68, 69]. These longitudinal studies would allow monitoring of disease activity and could

help to predict the patients’ outcome (relapse/remitting) or response to biological therapy.

The evaluation of cytokines can be performed in different conditions, and the information

collected could be significantly useful or not depending on many technical factors: the type,

specificity and sensibility of the assay used as well as the source of the medium in which the

cytokines were measured. About the media, note that cytokines are mediators active at

picomolar, and even femtomolar, concentrations that require a close control if present in the

blood to avoid deleterious effects. The severe clinical effects, and even death, of high

concentrations of cytokines in blood during a "cytokine storm" demonstrate the impact that can

have cytokines [70]. So, it could be possible to find low blood cytokine amounts during an attack

or a critical flare-up of an autoinflammatory immune disease, but during more quiescent, chronic

phases of the disease, the cytokines are often absent or found at trivial levels in the blood. They

may also be undetectable if their antagonistic molecules are present at high amounts.

Nonetheless, it has been suggested that baseline serum levels of cytokines or soluble cytokine

receptors may help predict the efficacy of biologics and select patients for cytokine-oriented

targeted therapies. For example, baseline levels of the soluble IL-6 receptor, measured by an

ultra-sensitive electrochemiluminescence assay in the presence of an immunoglobulin inhibiting

reagent to block heterophilic antibody interference, predicted clinical remission in many patients,

but not all, treated with the anti-IL-6 receptor tocilizumab [71]. One must also keep in mind that

the measurement of cytokines in the serum/plasma can sometimes be misleading. For example,

IFN-γ plays a pivotal role in systemic juvenile idiopathic arthritis-associated macrophage

activation syndrome, and its elevated circulating levels characterize the patients [72]. However,

upon an efficient treatment, serum CXCL9/MIG (Monokine Induced by Gamma interferon),

instead of serum levels of IFN- γ, correlates positively with disease activity. These observations

suggest that CXCL9 monitoring, rather than IFN-γ itself, could be useful for the evaluation of the

activity of this life-threatening complication of systemic juvenile idiopathic arthritis [73]. Also,

in the macrophage activation syndrome, serum IL-18 is chronically elevated, making it a

distinguishing biomarker among autoinflammatory syndromes. However, its source is entirely

derived from intestinal epithelia, at least in an experimental mouse model [74]. Therefore, these

are circumstances in which substantial serum cytokine levels can be detected, but their

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production is derived from a non-immune source. It is also useful to stress that limitations of

cytokine evaluation can be at the level of the causal agent responsible for diseases such as the

newly described monogenic autoinflammatory diseases related to protein misfolding,

endoplasmic reticulum response and proteostasis [75, 76].

Hence, the importance of evaluating the cytokine secretome must be objectively criticized: their

absence or trivial levels in plasma may not always be instructive, as opposed to their evaluation

in conditioned media from cells easily obtained and stimulated in vitro. However, it is not

necessarily obvious to determine, a priori, which cells should be tested and how they should be

stimulated, as many cell types are known to secrete cytokines. As examples, cells from

mesenchymal (i.e. fibroblasts, chondrocytes, osteoblasts, endothelial cells) and hematopoietic

(i.e. lymphocytes, monocytes, osteoclasts) origins produce cytokines and can all contribute to the

local pathology of inflammatory immune diseases [77-83]. However, the use of cells obtained

from tissues often requires relatively invasive techniques and labour-intensive purification steps,

procedures that can also affect the production of cytokines by the selected cells [84]. As a

primary and accessible approach, the use of peripheral white blood leukocytes should

definitively be considered to determine the cytokine secretome for patients suffering from

autoinflammatory and autoimmune diseases. As examples, patients with NOMID syndrome have

been reported with normal blood levels of cytokines while their PBMCs exhibited

overproduction of IL-1β, TNF, IL-3, IL-5 and IL-6 [85]. Another study examined the cytokine

profile in FMF patients to identify a specific cytokine signature and provide further evidence of

the cytokines that lead to the ongoing subclinical inflammation in these patients. On the one

hand, only IL-6 and TNF were enhanced in FMF patients’ serum, especially during crises,

highlighting the frustrating dataset information that can be obtained by serum alone. On the other

hand, the ex vivo PBMC stimulation from these patients, either in remission or during crises,

revealed the involvement of many other cytokines such as IL-1α, IL-1β and Th17-associated

cytokines (IL-17 and IL-22) and a decrease of Th1 (IFN-γ) and Th2 (IL-4) cytokines [86].

Moreover, PBMCs (and polymorphonuclear neutrophil leukocytes, especially during flare-ups

since their density could be modified, and some of them, the most active, will be collected

among PBMCs [87, 88]) are the immune cells which interact with tissues to lead to the

inflammatory and autoimmune process. Hence, it is also useful to remind that many immune

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cells recirculate from affected tissues and are present in PBMCs [18, 89, 90] (although this may

not be the case for certain resident cells), which is a major reason to evaluate the cytokine

secretome of PBMCs to learn about their inflammatory and immune behaviour.

Our recently published case report [91] highlights the autoinflammatory component of an

autoimmune disease, i.e. psoriatic arthritis (PsA) [92], as well as the difficulty of selecting an

appropriate therapeutic option. After several unsuccessful treatments with classical disease-

modifying anti-rheumatic drugs (DMARDs) and biologics, the patient greatly benefited from a

prospective investigation of the cytokine secretome. We demonstrated that the patient’s PBMCs,

and not the plasma, showed abnormal overproduction of a single cytokine, IL-6, as well as a

slight overproduction of IL-17 by stimulated T lymphocytes. Although IL-6 has been reported to

be involved in PsA, the treatment with the biologics tocilizumab (anti-IL-6R) or clazakizumab

(anti-IL-6) is not necessarily recommended [93, 94]. In light of the overproduction of IL-6 by the

patient's blood leukocytes, tocilizumab treatment was readily administered with a notable

improvement of about 60% of her overall condition and the normalization of the inflammatory

biological parameters. A genetic evaluation was also performed, and two genome-wide

association studies highlighted that the patient is a heterozygous carrier for the single nucleotide

polymorphism (SNP) rs33980500 in TRAF3IP2, the gene encoding the adapter protein TNFR

associated factor 3 interacting protein 2 (TRAF3IP2) [95]. Interestingly, variants of TRAF3IP2

are associated with susceptibility to PsA and psoriasis, and functional studies showed reduced

binding of TRAF3IP2 variant to TRAF6 [96]. The TRAF6-independent overproduction of IL-6

could be attributed to TRAF2 and TRAF5, as it was shown that both factors could transduce the

IL-17 signals leading to the stabilization of mRNA transcripts of cytokines such as IL-6 [97, 98].

Although the causal connection between the genetic variant and aberrant cytokine production

was not explored mechanistically in this patient, nor was the distribution among the different cell

types characterized, the demonstration of the abnormal cytokine secretion was necessary to guide

the treatment to the appropriate biologic, as the identification of a genetic variant alone was

insufficient to guide the treatment, making complementary strategies necessary [99].

This case report suggests that PBMCs' cytokine secretome may be useful to personalize the

appropriate biologic treatment. It also shows the importance of diagnosis of the

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autoinflammatory part of an autoimmune disease, even though it is not necessarily representative

of the inflammatory tissues, nor does it provide mechanistic insight into the disease. Many more

studies support the notion of evaluating the cytokines produced by PBMCs to personalize the

therapy in autoimmune diseases. For example, the comprehensive analysis of cytokine

concentrations in sera and anti-CD3-stimulated PBMCs of patients who have undifferentiated

arthritis progressing to RA revealed different cytokine profiles between patients refractory to the

DMARD therapy and patients responding to the therapy, suggesting that cytokine patterns may

be potentially used for the optimization of therapy introduction and monitoring [100]. Another

study revealed that the determination of IL-1β and the IL-1Ra/IL-1β ratio in the supernatants of

PBMCs cultured under resting conditions could be useful to predict the outcome of RA patients

undergoing treatment with methotrexate and may characterize a subset of patients that is more

responsive to IL-1-directed therapy [101]. Similarly, IL-1β measurement in whole blood cultured

with lipopolysaccharides was found to predict the response to anti-TNF therapies in RA [102].

However, one must keep in mind that the PBMCs’ cytokine secretome may not always be

informative to personalize the appropriate biologic treatment. For instance, the treatment

administered to the patient could sometimes have an impact on the pattern of cytokines produced

by ex vivo PBMCs [103]. Moreover, there are certainly cases where the secretome would be

similar to the ones of healthy donors, without any cytokines under- or over-secreted, and other

cases in which several cytokines would be abnormally secreted (unpublished personal case

report), making the choice of the appropriate biologics more tedious. Interestingly, the latter

condition could be the rationale for being more efficient by associating certain biologics

together, as already proposed [104, 105]. It is also important to mention that even in immune

disease in which the molecular mechanisms are rather well understood, such as

inflammasomopathies and interferonopathies, the treatment with biologics is often only partially

effective in some patients. In this sense, IL-1β blockade has proven to be effective in controlling

flares and joint inflammation in some patients with PAPA syndrome (pyogenic arthritis,

pyoderma gangrenosum, and acne), but does not seem to control for cystic acne, the second

cutaneous symptom of this inflammasomopathy [106]. Similarly, the treatment of patients with

SAVI (STING associated vasculopathy with onset in infancy) led to the reduction in febrile

episodes and skin lesions, along with improvement in lung function, but with incomplete type 1

IFN gene signature normalization in this interferonopathy [107]. Nonetheless, these observations

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could also be used as the rationale to establish the secretome of each patient to determine

whether a certain biologic is appropriate or not for them.

Another major issue relates to the permanent quality of samples, or in other words,

standardization of the methods used to allow rigorous comparisons and analyses. A specific

example could be at the level of cell condition to be studied. As for clinical tests, the use of

PBMCs freshly prepared, unlike cryopreserved PBMCs, would most likely help reduce or

control for technical variability and identify the biological difference between samples. In this

sense, variability in immune-based assays is often observed following PBMC cryopreservation

[108], including the detection of higher frequencies of cytokine-producing cells when ex vivo

versus cryopreserved PBMCs are stimulated with diverse antigens [109, 110]. The impact of

cryopreservation can be minimized with innovative cell culture approaches that use ex vivo

stimulation of blood cells with immune stimuli at the point-of-care and subsequent cytokine

quantification in stored supernatants, eliminating the need for cell cryopreservation and help

support harmonization of clinical studies and data sharing across multiple sites [111, 112].

Another point to consider in terms of standardization and comparison of data between

investigation centers will be at the level of percentages of various subsets present in PBMCs in

each sample to appropriately personalize the results and to lead to a better profile to adapt the

treatment. A pertinent detail is also at the level of efficacy of the detection antibodies in ELISA

used to measure cytokine concentrations in plasma, in particular for very low cytokine

concentrations such as those of type I IFN [34]. Hence, the standardization of the reagents used

to set up the ELISAs and similar immunoassays (multiplex technologies,

electrochemiluminescence, single-molecular array) using monoclonal antibodies [113-115] will

be of great importance to allow efficient comparisons of data without forgetting the possible

false-positive results in plasma/serum due to heterophilic antibodies (i.e. human anti-mouse

antibodies) possibly present in human plasma/serum [116, 117]). Regarding the cytokine

secretome and the complex functions of cytokines (pleiotropy, redundancy, the duality of

actions), it seems of significant importance to have the overall profile of cytokines involved in

each patient's disease. Notably, one could think that the exact knowledge of the cytokine

secretome of immune cells could drive more and more treatments towards a combination of

17

biologics to reach more rapidly and efficiently the clinical control of these debilitating diseases

[104, 105].

The evaluation of PBMCs' cytokine secretome should reflect the various stimulatory conditions

in which lymphocytes and monocytes can be found in vivo. On the one hand, innate mononuclear

cells express germline-encoded pattern recognition receptors that discern conserved motifs

expressed by pathogens. On the other hand, adaptive immune cells express receptors that

specifically recognize microbial antigens or peptides. Upon activation, these components of the

immune system converge to a standard set of signalling molecules, including nuclear factor

(NF)-κB, activator protein-1 (AP-1) and mitogen-activated protein kinase (MAPK), which drive

the production and subsequent secretion of cytokines and chemokines [118, 119]. As the ligands

recognized by these immune cell receptors have been extensively studied and are therefore well

documented [120-122], they can be used to stimulate in vitro the isolated blood cells and mimic

the in vivo activation processes. In this sense, ligands should trigger components of the innate

immune response, such as NOD-like receptors (NLRP3, NLRC4, Pyrin, Absent in melanoma 2

(AIM2), NOD1-2), Toll-like receptors (TLR1-9), RIG-like receptors (RIG-1/MDA5, STING), C-

type lectin receptors (DEC-205, mannose receptor, Dectin1-2, Mincle, DC-SIGN, DNGR-1) and

cytokine signalling (IL-1 family, IL-6, TNF, IFNs), as well as components of the adaptive

immune response, namely the antigen receptors of T (T-cell receptor) and B (B-cell receptor)

lymphocytes (Figure 2). Moreover, PBMCs can be obtained routinely in laboratory settings from

a venous blood specimen of the patients and their isolation, for subsequent activation, only

require a centrifugation step [123]. To control for experimental variances, a positive control

should be included, such as the phorbol myristate acetate (PMA)/ionomycin stimulation, known

to induce the secretion of a range of different cytokines in PBMCs [124], as well as blood cells

from healthy donors, ideally isolated the same day and stimulated with the same set of agonists,

in order to identify abnormally-secreted cytokines by the patient’s cells. Thus, this extensive set

of stimulations, either in isolated PBMCs or in whole blood, in combination with ELISA-based

assays, will, therefore, establish the cytokine secretome of PBMCs from

autoinflammatory/autoimmune patients and reveal abnormally-secreted cytokines that cannot

only be targeted by biologics but may potentially help establish a diagnosis. The latter, however,

remains to be determined.

18

Concluding remarks

Clinical symptoms of autoinflammatory or autoimmune diseases cannot systematically indicate a

specific diagnosis but only suggest the possibility of such a disease of which the complexity and

the numbers are increasing. The knowledge of cytokine secretome of PBMCs of these patients,

especially repetitive evaluation of this secretome over several months of evolution if possible,

could help target an efficient treatment, better understand the pathogenesis of the

autoinflammatory part of autoimmune diseases and may help clarify the diagnosis of each patient

as well, especially if this information associates immunological biomarkers with genetic and

epigenetic analyses. The establishment of the cytokine secretome of PBMCs could be

particularly useful when the diagnosis is difficult and could justify pursuing a genetic analysis, as

demonstrated by our recently-published case report. The cytokine secretome could also help

avoid the trial-and-error approach in patients that fail the classical immunosuppressive drugs and

first-line biologics. Also, since to date, the exact overall production of cytokines by ex vivo

PBMCs is unknown in most of the autoinflammatory and autoimmune diseases, the investigation

of cytokine secretome of patients with such diseases could bring an invaluable understanding of

the evolutive profile of these immune mediators that will open the best way of treatments.

19

Authorship P.E.P and M.P. researched, designed and wrote the manuscript. A.-L.C.N. revised and edited the

manuscript. All authors approved the manuscript for submission.

Acknowledgments We would like to thank Paul R. Fortin, MD, FRCPC (CRCHU de Québec-Université Laval) and

Ingrid Saba for critical review of the manuscript. The publication fees were sponsored in part by

a grant (#3623) from La Fondation du CHU de Québec. M.P. is a Junior 2 scholar from the

Fonds de recherche du Québec-Santé (FRQS).

Conflict of Interest Disclosure

The authors declare no conflict of interest.

20

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Figure Legends Figure 1: The classification of immunological diseases viewed as a continuum. McGonagle

and McDermott [7] proposed a continuum model in which immunological diseases lie on a

spectrum ranging from rare monogenic autoinflammatory diseases (e.g. hereditary fever

syndromes such as familial Mediterranean Fever and TNF receptor-associated periodic

syndrome) to rare monogenic autoimmune diseases (e.g. autoimmune lymphoproliferative

syndrome and immunodysregulation polyendocrinopathy enteropathy X-linked syndrome). This

model also encompasses polygenic autoinflammatory diseases (e.g. inflammatory bowel diseases

and gout) and polygenic autoimmune diseases (e.g. rheumatoid arthritis and systemic lupus

erythematosus) as well as mixed pattern diseases with evidence of acquired and

autoinflammatory components (e.g. psoriatic arthritis, ankylosing spondylitis and Behçet

disease). The continuum model provides a better understanding of the pathogenesis and

treatment options of self-directed inflammation.

Figure 2: Signalling pathways driving cytokine expression in peripheral blood leukocytes. The stimulation of different types of receptors of the immune response expressed by peripheral

blood mononuclear cells - monocytes, dendritic cells, natural killer cells, T and B lymphocytes -

leads to the activation of transcription factors and subsequent cytokine production such as TNF,

IL-6 and IFNs. Some cytokines like IL-1β and IL-18 need additional processing by the caspase-1

inflammasome to become fully mature. AIM2, absent in melanoma 2; AP-1, activator protein-1;

BCR, B-cell receptor; Ca2+, calcium ions; DAI, DNA-dependent activator of IFN-regulatory

factors; DAMP, damage-associated molecular pattern; DC-SIGN, dendritic cell-specific

intercellular adhesion molecule-3-grabbing non-integrin; DNA, deoxyribonucleic acid; DNGR-1,

dendritic cell natural killer lectin group receptor-1; IFN, interferon; IL, interleukin; IRF,

interferon-regulatory factor; JAK, janus kinase; MAPK, mitogen-activated protein kinase; MDA-

5, melanoma differentiation-associated protein 5; MR, mannose receptor; NFAT, nuclear factor

of activated T-cells; NF-κB, nuclear factor-κB; NLRC, NOD-leucine-rich repeat-caspase

activation and recruitment domain-containing 4; NLRP, NOD-like receptor; NOD, nucleotide-

binding oligomerization domain; PAMP, pathogen-associated molecular pattern; RIG-I, retinoic

acid-inducible gene I; ROS, reactive oxygen species; RNA, ribonucleic acid; STAT, signal

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transducer and activator of transcription; STING, stimulator of interferon genes; TCR, T-cell

receptor; TLR, toll-like receptor; TNF, tumor necrosis factor.