Features of Systemic Autoimmune Diseases

The mammalian immune system is made of an intricate set of cellular, chemical, and soluble protein mechanisms specialized in the protection of the organism from infections and tumors through the recognition and neutralization of pathogens or aberrant cells, without attacking the body’s own structures. In steady state, the immune system is tolerized to the antigens and the structures expressed by the body’s own cells and thus does not respond to elements that are expressed in endogenous tissues. All those peacefully coexist in a state known as self-tolerance. If the self-tolerance equilibrium is persistently unbalanced, this can ultimately result in development of autoimmune diseases, which can be described as the result of a sustained and persistent immune response against self-constituents. A variety of mechanisms have been described for the breakdown of tolerance supported by experimental models: failure in the deletion of autoreactive lymphocytes, central and peripheral tolerance malfunction, abnormal presentation of autoantigens, molecular mimicry, epitope spreading, or polyclonal lymphocyte activation (reviewed in [1]). A common feature of all autoimmune diseases is the presence of autoantibodies and/or self-reactive lymphocytes, chronic inflammation, and tissue destruction [2].

Nowadays, it is accepted that autoimmune reactions are part of the physiological functioning of the healthy immune system. In serum of normal individuals, the natural self-reactive antibodies are found at low concentrations and antigen avidity [3]. It is likely that natural autoantibodies are used by the organism to facilitate the clearance of senescent cells and cell-free autoantigens, and consequently, prevent the activation of cognate autoimmune responses. However, in the serum of patients with autoimmune diseases, high concentrations of IgG-switched autoantibodies are detected, which show high avidity for the antigen and somatic hypermutations of the variable region. These autoimmune response-associated autoantibodies are the product of a T-helper cell-dependent activation of B cells, which, in conditions of prolonged contact with the antigen, leads to clonal selection [4].

Regarding the target organs, autoimmune diseases can be classified into two groups: organ-specific and systemic autoimmune diseases (SADs). In SADs, the pathogenic antigens are widely expressed in the body, and therefore, many organs and tissues are targeted by the activated immune system [5]. The ubiquity of the autoantigens and the systemic nature of the resulting disorders may be the cause of many common signs and symptoms that accompany various SADs [6]. This group includes systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), primary antiphospholipid antibody syndrome (PAPS), mixed connective tissue disease (MCTD), systemic sclerosis (SSc), and Sjögren’s syndrome (SjS). Numerous mouse models have been employed to determine the genetic, molecular, and cellular mechanisms involved in the pathogeny of SADs. These models can be useful not only to elucidate disease mechanisms, but also to identify new genes associated to disease, to test new therapies, or to validate therapeutic targets [7,8,9,10].

Initial studies focused on the role of the adaptive immune system since primary abnormalities of B and T lymphocyte functions in SLE and other SADs were considered for a long time the likely basis of the autoimmune condition [11]. Recent advances in the understanding of the innate immune system have changed this first paradigm. Thus, it has been increasingly recognized that several components of the innate immune system, which detect pathogen patterns, play a key role in self-antigen recognition in autoimmune diseases [12, 13]. Both components of the immune system, innate and adaptive, are then involved in the physiopathology of organ-specific and SADs [14]. The cellular components of the innate immune system include monocytes, macrophages, dendritic cells (DC), and neutrophils, all being myeloid phagocytes. Although their differentiation pathways are not fully understood, they all originate from a common myeloid progenitor (see Fig. 1). These cells are involved directly in the pathogenesis of SADs, as antigen-presenting cells as well as acting as accessory cells through the secretion of soluble mediators like cytokines or chemokines (see Fig. 2). They play key roles in immune surveillance, host defense, and tissue repair, and their activity and differentiation fate can be differentially modulated by the tissue microenvironment and the characteristics of the hematopoietic niche in steady state or pathogenic conditions [15]. Most of the main relevant roles of myeloid cells in the pathogenesis of autoimmune diseases have been revealed in mice. In this review, we discuss the role of the main myeloid populations in the pathogenesis of SADs in patients, as well as the knowledge obtained using preclinical models.

Fig. 1
figure 1

Main steps and players in myelopoiesis. Graph depicting the still evolving view of the myelopoiesis pathways in mice and humans

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Fig. 2
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Myeloid cell involvement in SADs. Defective apoptotic cell clearance by myeloid phagocytes induces the accumulation of self-antigens in the tissues. DC take up these self-antigens and present them to autoreactive T and B lymphocytes in the presence of proinflammatory cytokines, inducing the active secretion of autoantibodies and the accumulation of pathogenic IC. Neutrophils recognize IC and, first, induce the release of ROS, cytokines, and proteases, and, second, die by NETosis. NET content activates pDC to secrete type I IFN, which in turn activates cDC and B lymphocytes, inducing antibody switching. Circulating monocytes migrate to the tissues and differentiate into tissue macrophages, which release ROS and mediate the differentiation of pathogenic resident cell types such as osteoclasts. Arrows, activated mechanism; whiskers, impaired mechanism

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Dendritic Cells

Human DC are a heterogeneous population composed by different subsets with differing phenotypic and functional features [16]: (i) plasmacytoid DC (pDC) can be differentiated from both lymphoid and myeloid precursors [17], producing huge amounts of type I interferon (IFN) after activation [18], and (ii) conventional DC (cDC) of myeloid origin. In steady state, human myeloid DC progenitors in the bone marrow (Fig. 1) originate two main specialized subpopulations characterized by the expression of BDCA-3 (CD141+) or, alternatively, BDCA-1 (CD1c+) [19]. During inflammation, an additional DC population of inflammatory DC (infDC) is generated in the tissues after differentiation of newly recruited monocytes [20]. Mouse DC present high homology with human DC [21]. Murine steady-state DC subsets can also be divided into pDC and cDC. cDC derive from a common precommitted precursor, the pre-cDC, that is dependent on FLT3L (Fig. 1) [22]. Murine cDC can be classified in CD8− and CD8+. Human cDC share key functional properties with their mouse counterpart, such as constitutive expression of MHC class II molecules, ability to process antigens and stimulate naïve T cells, as well as molecular signatures [23, 24]. cDC can be further divided into lymphoid organ-resident DC and migratory tissue DC. In this review, only classical myeloid DC (cDC or infDC) will be considered since pDC are beyond the scope of this review.

In healthy conditions, immature DC take up cell-derived self-antigens (autoantigens) in the tissues [25, 26]. DC present autoantigens to autoreactive T cells, but local immune suppressive factors induce tolerance through different mechanisms [27,28,29]. In the presence of danger signals, DC become mature and activated and initiate a maturation program optimizing their antigen presentation capabilities and their costimulatory activity [30, 31]. Mature DCs express high levels of MHC class I and II molecules, T lymphocyte costimulatory molecules and chemokine receptors, as well as an array of cytokines regulating adaptive immunity [32]. Depending on the nature of the environment, cDC can induce differentiation of Treg, Th1, Th2 or Th17 cells from naïve CD4+ T cells [20, 33]. cDC are also important for the promotion of the humoral responses [34] via the activation of specialized T follicular helper (TFH) cells or through the direct interaction with B cells [35, 36]. In addition, activated cDC produce high levels of B lymphocyte activation and survival factors, such as BAFF and APRIL, which have a key role in B lymphocyte differentiation and antibody production [37, 38]. The role of DC in inflammation and autoimmunity has been recently reviewed [39, 40].

DC in Mouse Models of SADs

Several animal models have shown that defects in different molecules acting as “eat me” or “find me” signals are involved in the SLE, RA, SSc, and SjS phenotypes, suggesting that a defect in apoptotic cell clearance by DC and other phagocytes can be driving the autoimmune condition (reviewed in [41]).

One of the main functions of DC is antigen presentation to T cells and the control of T cell differentiation. In general, DC ablation in mice leads to increased autoreactivity [42]. In the MRL-Fas/lpr mouse, the removal of DC drives a decrease in T cell expansion and in IgG and IgM autoantibody formation [43], but in inbred strains, DC depletion results in a normal development and number of total T and Treg cells [44]. These contradictory results can be explained by the background differences or by environmental factors, pointing out the relevance of the context on DC responses.

More specifically, gene ablation of different negative regulators of the immune activation in DC results in spontaneous autoimmune and/or inflammatory manifestations. For example, ablation of B lymphocyte-induced maturation protein-1 (blimp1) in DC induces an increased production of IL-6 and a preferential differentiation of follicular T helper cells (TFH) in vitro [45]. In a similar manner, at the same line, the deletion of the myeloid a20/tnfaip3 gene results in a polyclonal immune activation, spontaneous maturation of cDC, and an increase of cytokine production, accompanied by the development of an SLE-like disease [46]. A similar phenotype was observed after the depletion of the Src homology region 2 domain-containing phosphatase-1 (SHP1). The shp1-KO animals develop splenomegaly associated with more CD11c+ DC, increased numbers of Th1 cells, and an increased expression of CD86 and CCR7 in splenic cDC [47].

In RA, the main role proposed for cDC is the control of Treg differentiation. In mouse models of RA, injection of fully mature DC loaded with collagen prevents collagen-induced arthritis (CIA) after the induction of a Th2 shift [48]. Recently, it has been shown that the injection of immature cDC before collagen immunization suppresses the development of CIA by inducing a new subset of Tregs [49]. The maturation state of cDC has also been proposed to be important in RA development. Jaen et al. evaluated in the mouse CIA model the effect of cDC administration at different maturation states. LPS-stimulated DC are more effective than plasmid-stimulated DC in preventing the development of the disease and express indoleamine-pyrrole 2,3-dioxygenase (IDO), which may explain their better therapeutic effect. In both cases, the authors report an in vivo induction of Treg cells that appear first in lymph nodes and later in the spleen [50]. Immature DC (iDC) can expand and activate a novel regulatory population of CD49b+ T cells, with high immunosuppressive potential able to mediate protection against a systemic autoimmune disease [49]. The route of injection is also crucial in these experiments because the local injection of collagen-loaded mature cDC induces a local increase in the severity of arthritis [51]; however, cDC injected intravenously are able to prevent CIA [48].

Another key role of cDC is the production of B cell survival factors (see Fig. 2). Type I IFN increases the production of BAFF and APRIL by cDC, which are involved in the survival of autoreactive B cells, and this contributes to B cell differentiation and Ig class switching, which are important for generating pathogenic autoantibodies in SLE [52,53,54]. Deletion of the type I IFN receptor (ifnar) results in an ameliorated disease in different lupus-prone strains such as NZB [55], C57BL/lpr [56], (B6.Nba2 3 NZW)F1 [57], NZM2328 [58], and MRL/lrp [59]. More specifically, irak1 depletion in B6.Sle1 results in lower numbers of B cell blasts and activated CD4+ T cells [60]. In B6.Sle3, deletion of irak1 results in significantly reduced levels of anti-ssDNA and anti-double-stranded DNA (dsDNA) antibodies and a milder kidney pathology [60]. A similar approach was used by others and the deletion of stat4 in NZW derived mice (NZW2328) results in reduced levels of IFNγ but decreased survival rates and accelerated nephritis, even though levels of antibodies were low [61].

DC in SAD Patients

Flow cytometry and histologic analyses of DC subsets have shown a trend toward a reduced number of circulating DC in SAD patients, associated with an increase in the inflamed tissues [62,63,64,65,66]. Mature infDC have also been shown to be infiltrated in the synovial fluid of RA patients [20]. Mature cDC accumulate in the perivascular region of RA patients’ synovium, in association with T and B lymphocyte aggregates. These infiltrating cDC express the CCL20 receptor CCR6, which mediates the attraction of DC and Th17 cells to the tissues [67]. This finding suggests that a local maturation process is mediating the sequestration of DC in the leukocyte aggregates in the inflamed tissue in RA patients. In a similar manner, immune cell infiltrates of minor salivary glands of SjS patients contain typically macrophages and cDC [68].

Mature DC polarize naïve T lymphocytes into Th1, Th2, Treg, or Th17 through the secretion of different sets of cytokines. The accumulation of danger signals in the inflamed tissue stimulates and drives the DC to immunogenic or tolerogenic profiles. The release of cytokines that prime an improper autoantigen presentation leads to dysregulated autoreactive T and B lymphocytes that contribute to the physiopathology of autoimmune disorders. Mature cDC producing high amounts of Il-12 and IL-23 have been reported in the infiltrates of synovial tissues of RA patients, suggesting that these cells have a role in the polarization of pathogenic T lymphocytes [62, 66, 67]. infDC from synovial fluids of RA patients induce the secretion of IL-17 in naïve CD4+ T lymphocytes through the secretion of TGFβ, IL-1β, IL-6, and IL-23 [20]. In RA, IL-17 induces chondrocytes to secrete cartilage-degrading factors, and interferes with the synthesis of the cartilage matrix through the production of nitric oxide [69], or deregulating the RANK-RANKL (receptor activator of nuclear factor kappa-B ligand) pathway of osteoclast survival and differentiation [70].

As discussed before, suboptimal clearance of dying cells results in the accumulation of cell debris in the tissues and the ensuing local release of inflammatory signals [71]. Accordingly, a gene polymorphism in the mfge8 (milk fat globule-EGF factor 8 protein) gene, involved in phagocytosis mediating the interaction between phagocytes and dying cells [72], has been associated with SLE risk [73]. After antigen uptake in the tissues, mature DC migrate to the lymph nodes to activate antigen-specific T and B lymphocytes, in a process mediated by the CCR7 receptor and the chemokines CCL19 and CCL21 [74, 75]. Memory T cell activation by cDC can also occur within the inflamed tissues [76] in structured de novo formations with a follicular organization called ectopic lymphoid structures, where B cell activation leading to local production of pathogenic autoantibodies can be induced [77, 78]. The accumulation of cDC in autoimmune sites can be a consequence of the increased expression of chemokine receptors or their specific ligands in the tissue, or alternatively due to their defective migration to the draining lymph nodes from the inflamed tissue. Both mechanisms have been described in different SADs such as RA, SLE, and SjS [67, 79,80,81,82].

Monocytes and Macrophages

Monocytes have a critical role in innate immunity, not only as precursors of tissue macrophages and infDC but also through their function as phagocytes, antigen-presenting cells, and cytokine producers. Monocytes are produced in the bone marrow from hematopoietic stem cell precursors (see Fig. 1) and circulate in the periphery before migrating into the tissues, where they differentiate into different types of macrophages and DC [83]. Three major subsets of human circulating monocytes can be recognized by their surface phenotype: the classical CD14++CD16− monocytes, the nonclassical CD14+CD16++ monocytes, and an intermediate CD14+CD16+ population, considered as a transitional state between conventional and nonclassical monocytes [84]. After microbial stimulation, nonclassical monocytes are highly activated, become strong antigen-presenting cells, and produce high amounts of proinflammatory cytokines.

Macrophages are the main resident leukocytes in most tissues, differentiated in specialized phenotypes, e. g., Kupffer cells in the liver and microglial cells in the brain. Their numbers increase massively in inflammation and autoimmune diseases where they influence the normal cell turnover and tissue remodeling, facilitating the repair of injured sites [85]. Macrophages are known for their phenotypic heterogeneity, polarization, and plasticity. When the macrophages are recruited into the tissues, they become polarized, and generally, they can be classified as M1 macrophages, which are proinflammatory, and M2 macrophages, which are regulatory [86].

Monocytes and Macrophages in Mouse Models of SADs

In the autoimmune condition, the pathogenic roles of monocytes and macrophages are mainly due to alterations of immune complex (IC) recognition and clearance, nucleic acid recognition via toll-like receptors (TLR) signaling, and IFN signaling. Monocytes and macrophages, as well as other effector cells in the immune system, express cell-surface receptors specific for the Fc region of IgG (FcγR). They show differences in affinity according to the IgG subclass [87] and have a relevant role in autoimmune diseases [88]. The deposition in the kidney of autoantibodies in the form of IC and their interaction with FcγR is thought to trigger the local inflammatory response typical of SLE, leading to glomerulonephritis. Lostor-reduced expression of FcγRIIB results in development of lupus-like symptoms in the nonautoimmune C57BL/6 strain, with presence of autoantibodies and autoimmune glomerulonephritis, but this effect seems to be strain dependent [89]. In the NZB/NZW F1 mouse strain, direct activation of FcR in monocytes/macrophages is sufficient to initiate the response to glomerular IC deposit [90]. Mice deficient in FcγR do not develop proteinuria and inflammatory responses; however, the deposits of IgG and C3 are still present in the glomerulae [91,92,93,94]. In an interesting paper, Marino et al. identified a peptide able to bind to immunoglobulins and to interfere with FcγR recognition. Administration of this peptide to MRL/lpr mice results in a remarkable increase in the survival rate. Treated mice show lower IC deposition accompanied by a significant reduction in proteinuria [95]. These data demonstrate the relevance of FcR in controlling the kidney failure present in SLE, and blocking this receptor could be an attractive alternative to treat renal failure in the disease.

Another mechanism involved in lupus nephritis is the recruitment of monocytes and neutrophils mediated by type I IFN. In an experimental model of autoantibody-induced nephritis, the production of type I IFN by resident populations in the kidney seems to be responsible for tissue damage caused by deposition of autoantibodies. Increased levels of type I IFN aggravated the renal disease, whereas inhibiting IFN-I activity results in milder symptoms [96]. In the pristane-induced lupus model, a novel population of Ly6Chigh macrophages has been described as the main producer of type I IFN independently of DC activation [97]. Ly6Chigh monocytes from the bone marrow go into the circulation and then to the peritoneal cavity where they accumulate. A striking correlation between the numbers of Ly6Chigh monocytes and the production of autoantibodies is also observed. Monocyte depletion results in a decrease in type I IFN and IFN-induced gene expression, though the systemic depletion of DC has little effect. The expression of TNFα also diminished upon CD11b+Ly6Chigh monocyte depletion, whereas the expression of IL-12 does not change significantly. These results support the possibility of production of type I IFN by immature monocytes independently of DC in lupus [97].

To elucidate the role of complement in this model, the same authors designed new experiments in two different knockouts: C1qa (BALB/C and C57BL/6 strains) and C3 (BALB/c). Surprisingly, C1qa−/− mice develop lower titers of circulating autoantibodies and milder arthritis compared with the controls. Two months after pristane injection, a decrease in the number of CD11b+Ly6Chigh monocytes in peritoneal exudates was detected in C1qa−/− mice; conversely, the number of the circulating population was higher. In vitro, peritoneal macrophages from C1qa−/− BALB/c mice injected with pristane produce less CCL3, CCL2, CXCL1, and IL-6 after TLR7 stimulation in vitro, but after stimulation of TLR3, TLR4, and TLR9, the levels of cytokines/chemokines are similar to WT animals. Deletion of other complement components, such as C3, does not affect the chemokine/cytokine production in the same conditions [98]. Based on these data, we can conclude that C1qa has an important role in the recruitment of circulating monocytes to the peritoneum in the pristine-induced lupus model.

Elevated levels of cytokines and chemokines in tissues also contribute to SLE development and can lead to renal leukocyte infiltration and tissue damage. The presence of leukocytes in renal infiltration is usually associated with poor prognosis in SLE. During experimental lupus nephritis, F4/80hi cells expressing high levels of CD11b, CD80, CD86, MMP2, MMP14, Ikkε, CXCL13, and IL-10 are a major renal source of proinflammatory cytokines and chemokines [99]. In NZB/W mice, nephritis onset is associated with a specific renal macrophage/DC signature. Renal F4/80hi/CD11cint macrophages are located throughout the interstitium, whereas F4/80lo/CD11chi DC accumulate in perivascular lymphoid aggregates. CD11b+/CD11chi/F4/80lo cells appear in large numbers in lymphoid aggregates during nephritis [99] and disappear upon remission. A new type of renal F4/80hi/CD11cint macrophage has been described in the kidney with a Gr1lo/Ly6Clo/VLA4lo/MHCIIhi/CD43lo/CD62Llo phenotype different from that described for inflammatory macrophages [100].

High levels of expression of two ligands for CCR1, CCL3, and CCL5, in association with mononuclear phagocytes and T cell infiltration, have been reported in NZB/W mice, as well as in other models of SLE and in human lupus nephritis [101,102,103]. In mouse models of lupus nephropathy, the expression of CCR1 on myeloid and some subsets of T cells seems to guide them to inflamed target organs such as the kidney. In NZB/W mice, CCR1 inhibition ameliorates the progression of lupus nephritis [104]. MRL(lpr/lpr) mice treated with the CCR1 antagonist BX471 show a reduced renal expression of CCL2, CCL3, CCL4, and CCL5 and the chemokine receptors CCR1, CCR2, and CCR5, together with reduced kidney fibrosis. However, this treatment has no effect on the levels of serum anti-dsDNA autoantibodies, proteinuria, or glomerular injury [105]. Short-term treatment with the orally available CCR1 antagonist BL5923 resulted in lower numbers of T cells and macrophages in the kidney infiltrates [104]. At longer times, CCR1 antagonist administration results in a minor kidney accumulation of effector/memory CD4+ T cells, Ly6C+ monocytes, and both M1 and M2 macrophages in MRL-lpr mice. The tissue damage is reduced resulting in a delayed proteinuria and increased survival [105]. In transference experiments done in the NZB/WF1 model, it has been reported that splenic T, B, and myeloid cells from nephritic mice migrated into noninflamed syngenic kidneys [102]. When the transfer was done to chronically inflamed kidneys, the process was improved, suggesting that this process could be autoregulated with a loop between kidney signaling and circulating leukocytes.

The deficiency on interferon regulatory factor 4 (IRF4), a transcription factor required for M2 macrophage polarization [106], also inhibits TLR signaling through its binding to MyD88 [107]. As a consequence, irf4 deficiency enhances the activation of antigen-presenting cells and the production of NF-κB-dependent proinflammatory cytokines in the lupus-prone B6lrp mice but protects the animal against IC deposition in the kidney and the resulting glomerulonephritis [108].

Macrophages are also a relevant population in the control of other SADs as RA in human and mouse models. In this disease, the most relevant role of the macrophages is the production of cytokines that control osteoclast activity (Fig. 2). Th1 cytokines, such as IL-12 and IFNγ, and Th2 cytokines, such as IL-4 and IL-10, are inhibitory for osteoclastogenesis [109,110,111,112]. It is also relevant in the production of NO by synoviocytes and macrophages that induces degeneration of chondrocytes. Other cytokines such as colony-stimulating factor 1 (CSF-1) and its receptor, CSF-1R, play an important role in regulating tissue-resident macrophages and osteoclasts. The expression of CSF1-R increases during CMP differentiation to macrophages (Fig. 1) and CSF-1R downstream signaling regulates macrophage survival, proliferation, differentiation, and chemotaxis [113]. In two different RA models, such as the CIA model and the passive serum transfer, the blockade of CSF-1R abrogated cartilage damage, bone erosion, and systemic bone loss. In both cases, this effect was associated with depletion of osteoclasts. A significant reduction in inflammation was also observed that was accompanied by the absence of synovial macrophages and a reduction of the number of splenic monocytes, pointing out the relevant role of CSF-1R in controlling these populations in RA [114].

SjS animal models show a predominance of CD4+ T lymphocytes infiltrated into lachrymal and salivary glands. The presence of macrophages in the infiltrates has been detected but their pathogenic role is not well defined yet, even though it is known that they are important players [10, 115]. In NOD mice, macrophages and DC initiate the infiltration into the salivary glands that will develop into a lymphocytic focus. Therein, M1 and M2 macrophages are detected together with B and T cells [116]. The role of macrophages in SjS pathogenesis has been investigated by Zhou et al. using a knockout mouse model for the autoimmune regulator (AIRE). These knockout mice present a multiorgan autoimmune disease, including an exocrinopathy affecting the salivary and lacrimal glands [117, 118]. In the absence of AIRE, F4/80+ macrophages accumulate in the cornea. Subconjunctival injection of clodronate liposomes depletes macrophages locally with no effect on CD11c+ DC and improves corneal epitheliopathy, hyperplasia, and stromal fibrosis. In AIRE KO mice, macrophages appear to function locally, downstream of CD4+ T cell activation and infiltration. Clodronate systemic administration does not improve the ocular epitheliopathy but results in an improvement in tear secretion and decreased damage to lachrymal glands [119]. Thus, even if CD4+ T cells are the main population in the infiltrates and primary effectors in the development of the pathogenesis, macrophages seem to have a relevant role in the development of the complete SjS phenotype. In the NOD/B10-H2 b strain after prophylactic treatment with cobra venom factor (the complement-activating protein from cobra venom that functionally resembles C3b), animals failed to develop salivary dysfunction and showed reduced levels of leukocyte infiltration, reduction of antinuclear autoantibodies, and major alterations in the B lymphocyte profiles [120]. The role of complement has also been studied in the C57BL/6.NOD-Aec1Aec2 SjS mouse model. In this case, the deletion of C3 resulted in a decrease in clinical signs. C3 KO animals presented reduced acinar cell apoptosis, reduced levels of caspase-3, lack of leukocyte infiltration of submandibular glands, and reduced synthesis of pathogenic autoantibodies. The glandular architecture and retention/secretion of saliva were normal [121]. RNA expression microarray studies have been carried out in lachrymal glands of NOD mice comparing them with age-matched BALB/c mice. The results showed an upregulation of cathepsins and proinflammatory factors including TNFα, IL-6 and IL-1β [122]. In C57BL/6.NOD-Aec1Aec2 mice, caspase-11, expressed primarily in macrophages and DCs, was significantly upregulated at 8 weeks of age, but not caspase-9 [123]. The upregulation of caspase-11 in the submandibular gland before disease onset is apparently associated with the enhanced transcriptional activity of the signal transducer and activator of transcription 1 (STAT1) gene [124]. In general, the presence of elevated levels of proinflammatory cytokines in the submandibular gland enhances IFNγ production by epithelial cells, resulting in further activation of macrophages [124].

It is also worthy to mention the role of macrophages in SSc. Macrophages are a potent source of reactive oxygen species (ROS). ROS have multiple effects including DNA oxidative damage and unbalanced oxidative stress, which has been implicated in the pathogenesis of scleroderma. There are increased numbers of macrophages at the early stages of fibrosis, and they release proinflammatory and fibrogenic mediators, such as TGFβ and PDGF [125]. A high number of macrophages have been detected in the skin of Scl-GVHD [126] and bleomycin-induced mouse models [127]. In the bleomycin model, TGFβ is produced by fibroblasts and infiltrating cells that are predominantly comprised of macrophages at the sclerotic stage [128]. CCL2 and its receptor CCR2 are upregulated in dermal fibroblasts and inflammatory cells from both SSc bleomycin-treated mice as it happens in patients [129]. In the CCL2-deficient mice, skin fibrosis was diminished even after the bleomycin treatment [130]. In the Scl-GVHD model, populations of monocytes/macrophages (CD11b+/2F8+) and CD3+ T cells of donor origin are the main components of the skin lesion. There are also high levels of CCL2, IFN-inducible chemokines, VEGF, and adhesion molecules in the skin [131].

Monocytes and Macrophages in SAD Patients

One of the major functions of blood monocytes is the elimination of opsonized microorganisms and apoptotic debris by phagocytosis or receptor-specific endocytosis through pattern-recognition receptors (PRR) [132]. Among them, C1q mediates the recognition of a wide variety of plasma proteins and pathogen molecules [133, 134], ensuring uneventful removal. It has been shown that the deficiency in the C1q protein is associated with a risk of developing SLE and RA [135, 136], suggesting that the role of C1q in the clearance of microbial elements and self debris could be crucial for preventing the induction of pathogenic autoantibodies [137]. Circulating monocytes from SjS patients release spontaneously higher amounts of the two B lymphocyte-stimulating cytokines IL-6 and BAFF [138] and show high levels of phosphorylation of STAT5, correlating with serum IgG levels and anti-SSB/La autoantibody titers [139]. However, they show an impaired capacity of phagocytosis of apoptotic cells [140].

A significant role for monocyte activation in PAPS-mediated thrombogenesis has been suggested. From a proteomics analysis of monocytes from PAPS patients with thrombosis, a differential expression of annexin I and annexin II, as well as RhoA, Nedd8, and Hsp60 proteins, has been observed [141]. Circulating antibodies and autoantibodies from PAPS patients activate monocytes through TLR2 and CD14, inducing the expression of ROS and the secretion of tissue factors [142, 143]. They also induce the overexpression of TLR8 and its translocation from the endoplasmic reticulum to the endosomal compartment, sensitizing monocytes to TLR8 ligands [144].

SSc patients have a higher proportion of CD14+ monocytes in the blood, showing an activated phenotype [145]. These activated monocytes overexpress both CD169, a macrophage marker induced by type I IFN, and CD204, a marker for activated profibrotic M2 macrophages [146]. Moreover, LPS stimulation of SSc circulating monocytes increases CD163 expression compared to monocytes from control individuals [147].

SLE macrophages are unable to clear efficiently apoptotic cells and show an altered proinflammatory status characterized by an overproduction of inflammatory cytokines, such as type I IFN, TNFα, and IL-6 [148, 149]. They show enhanced antigen presentation capacity and are primed for activation, leading to a skew toward autoimmunity [150]. In this inflammatory context, SLE monocytes and macrophages present self-antigens to autoreactive T lymphocytes instead of inducing peripheral tolerance after phagocytosis of apoptotic cells [148]. Interestingly, CD68+ mononuclear phagocyte infiltration in the kidneys of lupus nephritis patients is associated with poor prognosis [151,152,153].

Macrophages produce many proinflammatory cytokines and chemokines in the synovial tissue of RA patients, contributing to cartilage and bone destruction [154]. Indeed, infiltrating macrophage numbers constitute a biomarker for disease severity, as well as a predictor of the response to therapy [155]. It has been reported that there is a positive correlation between the number of infiltrated macrophages and the degree of joint erosion [156]. The polarization of RA synovial tissue macrophages depends on the stage of the rheumatic inflammation. Actually, patients with highly active RA show a prevalence of the M1 phenotype. On the contrary, macrophages of RA patients with low disease score or in clinical remission show an M2 phenotype [157], and furthermore, glucocorticoid treatment induces an M2 state [158]. The infiltration of macrophages into the labial salivary glands of SjS patients correlates with the biopsy focus score [159]. Additionally, patients with SjS show higher expression of IL-18 in the infiltrated macrophages, with a positive correlation with salivary gland enlargement [68].

Skin infiltrates of SSc patients are composed mainly of T lymphocytes and macrophages. Among them, infiltrated CD163+ macrophages seem to be the main source of CCL19, a chemokine strongly correlated with vascular markers, suggesting a role of CCL19 in the recruitment of macrophages to the inflamed SSc skin [160]. In terms of phenotype, it has been reported that there is overexpression of TLR4, CD14, and MD2 in the skin of diffuse SSc patients. The expression of these genes correlates with progressive skin disease [161], suggesting that these markers can be used for the monitoring of skin disease progression. CD14 is mainly expressed by macrophages, although it can also be expressed at lower levels by cDC and neutrophils. In addition, dermal macrophages of SSc patients acquire a profibrotic phenotype after stimulation with IL-13 [162, 163]. Microarray analysis of lung samples of patients of SSc-associated interstitial lung disease shows a unique gene signature compared with other similar lung diseases. Many genes of this specific signature correspond to alveolar macrophage activation and fibrosis [164, 165]. Studies of expression profiles of bronchoalveolar lavages of SSc patients with lung inflammation describe the induction of markers of alveolar macrophage activation [166], together with a consistent increase in the expression of CCL18 transcripts [167]. This result is in agreement with the high levels of serum CCL18 in SSc patients, related with lung involvement [168,169,170]. In addition, it has been shown that circulating monocytes and alveolar macrophages of SSc patients of interstitial lung disease responded more intensely to LPS stimulation [147, 166].

Neutrophils

Neutrophils have key roles in the control of infectious agents through their capability to quickly migrate from the circulation to the infected tissues in response to regulatory or chemotactic signals [171]. Once they arrive at these sites, they turn into “primed” neutrophils, recognizing and destroying the invading pathogens using a wide variety of degrading enzymes contained in their granules, in addition to their ability to generate ROS [172]. Using these arms, neutrophils have the highest killing activity among the immune cells. Primed neutrophils extend their lifespan and promote inflammation using chemokines and cytokines that attract other actors of the immune system, and regulating almost every element of the inflammatory response [173]. When the infection is resolved, they die by apoptosis [174] or NETosis, through the release toward the extracellular milieu of granule-derived protein-decorated chromatin forming neutrophil extracellular traps (NETs) [175, 176]. High titers of autoantibodies against dsDNA, histones, or anti-citrullinated protein antibodies (ACPA) are hallmarks of SAD patients. Since the proteins associated with the DNA in the NETs include citrullinated histones and proteins with altered immunogenicity after posttranslational modifications such as oxidations, neutrophils can also be a source of autoantigens through degranulation or NETosis [177,178,179].

In some conditions, neutrophils can also infiltrate tissues and become improperly activated in sterile tissues via deposed IC [180], and secrete the content of their granules, attacking host tissues if local detoxification pathways become overburdened. As a consequence, the connective tissues are dissolved and normal cells destroyed [181]. Besides their direct tissue damage induction, neutrophil-derived regulatory factors also organize a sterile inflammatory response [182]. Neutrophil-secreted cytokines have been shown to contribute to the deregulation of the immune responses in several SADs [173]. Other neutrophil functions have been shown to be deregulated in several SADs as shown in Table 1 and detailed below.

Table 1 Role of myeloid cells in systemic autoimmune diseases
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Neutrophils in Mouse Models of SADs

Strong evidence about the important role of neutrophils in SLE pathogenesis comes from in vivo depletion experiments [184]. The depletion of the neutrophil population in lupus-prone autoimmune B6.Faslpr/JTnfrsf17−/− mice, deficient in a BAFF receptor, results in a reduction in autoantibody titers, serum IFNα and BAFF, T cell activation, as well as high numbers of splenic germinal center B cells and plasma cells. In this strain, high production of BAFF by neutrophils may help to drive the selection and survival of autoimmune B cell clones that produce self-reactive antibodies, such as anti-dsDNA antibodies [184]. Interaction between BAFF, T cells, and IFNγ has also been proposed in the Lyn-deficient autoimmune mouse model. Lyn−/− mice present a ∼30–50% reduction in mature B cell numbers, and lyn −/− myeloid cells are hyperresponsive to engagement of surface integrins, showing increased secondary granule release [205]. Scapini et al. have described a population of hyperactivated myeloid cells in these animals that produces high levels of BAFF, that activates T cells to release high levels of IFNγ. Administration of anti-BAFF monoclonal antibody reduced disease development in Lyn−/− mice and a similar effect was observed with the genetic deletion of IFNγ [185].

Other important mechanism by which neutrophils drive autoimmune responses is through the release of ROS, proteases, and proinflammatory cytokines (Fig. 2). In the MRL/lpr mouse, blockade of mitochondrial ROS production has recently been reported to be sufficient to block NETosis in vitro, reducing disease severity and type I IFN responses [186]. The KO of the NADPH oxidase nox results in increased lupus disease symptoms in this particular strain [206]. However, the role of NOX in immune responses is not all clear since in patients with chronic granulomatous disease and lack of functional NOX, a proinflammatory phenotype has been observed [207].

The relevance of NET formation in lupus disease has been tested recently in animal models. Injection of netting neutrophil cell lines to wild-type mice does not result in the development of lupus disease. Although it is unclear if those injections could mimic the in vivo NET formation and prime the activation of TLR signaling, IgG and IgM antibody levels were increased [187]. Another key process in NET formation in vivo is the citrullination of histones by peptidyl arginine deiminase 4 (PAD4). Neutrophils from MRL/lpr and NZ2328 mice demonstrate accelerated NET formation compared with controls and an accelerated NET formation [188, 189]. In MRL/lpr inhibition of PAD1, PAD2, and PAD4 using the Cl-amidine inhibitor markedly improves endothelial function and reduces proteinuria and IC deposition in the kidney while protecting against skin disease [188]. In NZM, the same treatment inhibits NET formation in vivo and significantly alters circulating autoantibody profiles and complement levels while reducing glomerular IgG deposition [189].

In RA, neutrophils also have a critical role in the initiation and maintenance of the disease. Neutrophils are abundant in murine autoimmune arthritis and contribute to the pathogenesis through the release of cytotoxic products and immunoregulatory mediators. In addition, neutrophils may promote autoimmunity by formation of NETs and the associated promotion of anticitrullinated protein/peptide antibodies [182, 208]. Interestingly, neutrophil-depleted mice are completely resistant to the disease-inducing effects of K/BxN serum transfer [209]. In CIA models, it has also been proven that they have a critical role in initiating and maintaining the inflammatory responses. In a similar way to what happens in patients, there is also a prominence of neutrophil recruitment in RA models [209,210,211]. It has been demonstrated that neutrophils participate in their own recruitment in murine arthritis through C5aR and FcγR signaling [212]. Once in the joints, neutrophil activation by IC promotes IL-1β production, which stimulates synovial cells to produce chemokines, amplifying the neutrophil recruitment into the joints [212]. Neutrophils infiltrating the synovial membranes and joints in rats with arthritis upregulate cathelicidins [213], antibacterial peptides with potent proinflammatory and immunomodulatory activities [214].

Recent evidence indicates that the inflammatory loops initiated by the molecules externalized in NETs may be key in arthritis development [215]. As it was mentioned before, citrullination of histones by PAD4 is a key step in NET formation. PAD4 mRNA, absent from healthy synovium, is transcribed and translated by neutrophils infiltrating synovial tissue during inflammation. As a consequence, several synovial proteins are citrullinated in this compartment [216]. Of interest, the PAD inhibitor CI-amidine mitigates collagen-induced arthritis and decreases the clinical disease score [190]. However, no abrogation of disease severity was observed in the PAD4 knockout mice using the K/BxN serum transfer model of arthritis [217].

Granulocyte-colony stimulating factor (G-CSF) has also been found to be a key player in arthritis models, participating in the interactions between hematopoietic cells through the control of myeloid cell numbers and activation [218]. Neutrophil depletion or reduction of their G-CSF production also inhibits disease development in the CIA arthritis model [191]. This cytokine is also required for neutrophil recruitment in the K/BxN serum transfer arthritis model [192]. In this line of research, antibody blockade or knockout of key neutrophil signaling receptors, such as CXCR1 and CXCR2, ameliorates disease signs in an antigen-induced model [193, 194]. A similar effect of reduction in disease progression was observed in the CIA model with ablation of C5aR [195], involved in neutrophil recruitment [212]. In the K/BxN serum transfer-induced arthritis model, an important role of FcγR has also been demonstrated. Expression of human FcγRIIa on neutrophils in mice that lacked their own results in the restoration of susceptibility to K/BxN serum induced RA, neutrophil recruitment, synovitis, and bone destruction [219]. Other molecules involved in the recruitment of pathogenic neutrophils are L-selectin [196], IFNγ, [197, 198], and IL-17 [199]. Neutrophil production of IL-17 has also been pointed to as an amplifier of arthritis in the K/BxN model [200]. Neutrophil activation following recognition of early IC in the joint may also lead to changes in vascular permeability, which further promotes IgG deposition [220]. The production of other effectors, such as ROS, is also important for the induction of the disease. In summary, we can conclude that decreased disease activity and joint destruction directly correlates with lower influx of neutrophils to joints and less neutrophil activity.

Production of ROS by neutrophils seems to also be a concern in SSc pathogenesis [203]. Repeated injections of hypochlorous acid, a product of neutrophil burst, induced skin and lung fibrosis as well as anti-DNA topoisomerase 1 (Scl70) antibody production, mimicking the diffuse form of SSc in patients and proving the relevant role of ROS in SSc [203, 204].

Neutrophils in SAD Patients

Neutropenia is found in a significant proportion of SLE patients as reported in [221, 222]. Circulating neutrophils of SLE patients display abnormal features, such as impaired phagocytic activity [223] and lower recognition by the C1q-mediated apoptotic cell clearance [224]. The lower production of ROS by circulating neutrophils from SLE patients with more severe symptoms indicates that these cells are not primed but show a skewed phenotype [225]. On the other hand, enriched numbers of low density granulocytes (LDG), with an activated phenotype but morphologically similar to immature cells, are characteristic in the blood of these patients [226, 227]. The number of circulating LDG correlates with dsDNA-specific autoantibody titers and disease severity [228]. The enhancement of NETosis activity in LDG and neutrophils from SLE patients is well established. It has been hypothesized that neutrophil death induces the type I IFN production by pDC characteristic of SLE [229], facilitating the uptake of extracellular DNA by pDC and their activation [178]. IFN and IC trigger the activation of neutrophils, inducing again their NETosis in a self-amplifying process [230]. Several studies have reported the finding of neutrophils in the kidney biopsies of lupus nephritis patients [230,231,232,233], and tissue NETosis has been correlated with higher titers of anti-dsDNA autoantibodies [230]. Altogether, these data support the notion of neutrophils having a key role in the pathogenesis of SLE [234].

In healthy individuals, blood neutrophils need to be primed in order to migrate to the tissues and become active. In RA patients, circulating and infiltrated neutrophils have a longer lifespan [235] and show an activated phenotype [236], together with activation of the NF-κB pathway [237], increase of their chemotactic capacity [238,239,240], high phagocytic activity [241], and enhanced ROS production [242] in response to IC [243]. This phenotype participates actively in the damage of the synovial joints [182]. The process consisting in the adherence of activated neutrophils to IC in the synovial fluid, causing degranulation and liberation of ROS and collagenases [244], has been called “frustrated phagocytosis” [245]. The phenotype of synovial neutrophils of RA patients is quite similar to tissue macrophages, in terms of secretion of a wide variety of proinflammatory cytokines and chemokines [174], thus facilitating the delay in the neutrophil apoptosis induction. Recent reports suggest a role of neutrophil NETosis in the joint damage in RA [182], since anti-citrullinated protein antibodies (ACPA) are characteristic of erosive RA, and NETs contain citrullinated histones [179]. Spontaneous NETosis of neutrophils in culture is higher in RA compared with controls, and they have more nuclear citrullinated histone H3 [246]. Interestingly, antibodies specific for citrullinated vimentin are associated with the severity of RA [247]. Moreover, neutrophils from healthy donors bearing the T allele of the RA risk-associated gene ptpn22(C1858T) have a high migration capacity, superior ROS production, and enhanced NET release [248], indicating that the neutrophils could be acting in the very first steps of the pathogenic processes of the disease.

The information about the role of neutrophils in the physiopathology of other SADs is less complete compared to SLE and RA. For instance, there are some pieces of evidence pointing to a role of NETosis in the pathogenesis of PAPS. Similar to SLE, the sera of PAPS patients show a decrease in the NET-degrading activity [249]. In line with this finding, the sera of these patients also have high levels of cell-free DNA and NET components, and the circulating neutrophils have high spontaneous NETosis activity [250]. A LDG population has been also described in the blood of PAPS patients [251]. Other neutrophil functions, such as ROS generation, are skewed in neutrophils of SSc patients [252, 253].

Concluding Remarks

As summarized in Table 1, scientific evidence pointing to a key role of the myeloid cells in the pathogenesis of SADs is abundant and strong. Dendritic cell alterations in immune diseases include presentation of self-antigens to autoreactive T cells, increased secretion of proinflammatory cytokines, and promotion of autoantibody production in B cells. DC also act through the control of T cell differentiation and activation. Monocytes and macrophages are important cytokine producers able to control migration of other populations to the inflamed tissues and remodeling processes such as condrogenesis and osteoclast activity. They respond to the IC deposit and produce proinflammatory cytokines and chemokines participating in tissue damage in SADs. Finally, neutrophils release ROS, proteases, and proinflammatory cytokines that act as danger signals. Netting neutrophils release intracellular modified antigens promoting the induction of pathogenic autoantibodies.

According to these data, our challenge in the next few years is to better dissect the immunopathological mechanisms underlying these disturbances in order to define specific cell subsets or proteins that can be potential targets for drug development.

cDC conventional dendritic cells, CIA collagen-induced arthritis, DC dendritic cells, IC immune complex, infDC inflammatory dendritic cells, IFN interferon, pDC plasmacytoid dendritic cells, RA rheumatoid arthritis, ROS reactive oxygen species, SADs systemic autoimmune diseases, SjS Sjögren’s syndrome, SLE systemic lupus erythematosus, SSc systemic sclerosis