The Ca²⁺/calmodulin/calcineurin pathway regulates the activity of the transcription factors of the nuclear factor of activated T cells (NFAT) family. The NFAT family encompasses five individually encoded members. NFAT1 (also called NFATc2 or NFATp), NFAT2 (NFATc1 or NFATc), NFAT3 (NFATc4), NFAT4 (NFATc3 or NFATx) 23 , and NFAT5 (also called TonEBP or OREBP) 24 . All NFATs share a similar DNA-binding domain, and are modulated by calcineurin, a calcium, calmodulin-dependent serine/threonine protein phosphatase, consisting of a catalytic subunit calcineurin A (CnA), and a tightly associated Ca²⁺-binding subunit, calcineurin B (CnB) 25 . NFAT5, which differs in its structure from the other NFATs, is not regulated by calcium, but is activated in response to osmotic stress. NFATs are maintained in an inactive state in the cytosol of resting cells. Upon the stimulation of intracellular Ca²⁺ influx, calmodulin is activated and dephosphorylates the phosphorylation motifs from the N-terminus of NFATs, allowing NFATs to translocate to the nucleus where they collaborate with other transcription factors, such as AP-1, to induce gene transcription 26 . Calcineurin inhibitors, which inhibit the phosphatase activity of calcineurin and the nuclear translocation of NFATs, are currently used to prevent graft rejection in transplant recipients. Impaired activation of NFATs will prevent the transcription of cytokine genes, including IL-2, in activated T cells 26 . However, in addition to their major functions in lymphocytes in adaptive immunity, new roles of NFATs in innate immunity have been identified in myeloid cells 27 .

Dectin-1, which plays a key role in the recognition of pathogenic fungi, is a β-glucan receptor belonging to the C-type lectin receptors, and is activated by zymosan, a cell-wall constituent of Candida species 28 29 30 . Upon ligand activation, dectin-1 cooperates with TLR2 to stimulate NF-κB and regulate cytokine production 31 . Dectin-1 alone was shown to induce phagocytosis and Src and Syk kinases-mediated induction of reactive oxygen species (ROS) via the activation of NFAT in macrophages and DCs 32 . Dectin-1 also regulates the induction of members of early growth response (Egr) family, cyclooxygenase-2 (COX2), and IL-2, IL-10, and IL-12 p70 productions by DCs stimulated by zymosan 32 . CD14 was also shown to induce Ca²⁺ influx and NFAT activation causing apoptosis of differentiated DCs, but not macrophages, stimulated by LPS 33 . NFAT2/c1 activated by the receptor activator of NF-κB (RANKL) was also shown to play a key regulatory role in the terminal differentiation of osteoclasts 34 .

Recently, Fric et al. 12 demonstrated that NFAT1/c2 acts as a negative regulator of myeloid lineage development. These authors showed that inhibition of calcineurin/NFAT signaling increases the number of myeloid progenitors, and that the two calcineurin inhibitors CsA and FK506 antagonized the development of DCs induced by the Fms-related tyrosine kinase 3 ligand (Flt3-L). These findings demonstrated that calcineurin/NFAT signaling contributes to maintenance of innate immune homeostasis 35 .

A number of studies have provided indirect and more direct evidence that NFAT/calcineurin pathways interfere with the regulatory mechanisms of innate immune defences. Calcineurin inhibitors or knockdown of calcineurin mRNA expression activate NF-κB and TLR-mediated MAPK pathways in macrophages, while over-expression of a constitutively activated form of the CnA subunit inhibits TLR- activated pathways 36 . The development of selective inhibitors of NFAT, such as VIVIT, a high-affinity calcineurin-binding peptide selected from combinatorial peptide libraries based on the calcineurin docking site of NFAT 37 , and the more recent cell permeable inhibitor of NFAT, 11R-VIVIT 38 have proved to be useful tools to analyse in vivo the functions of NFAT. 11R-VIVIT and FK506 significantly inhibit LPS and LPS plus IFN-γ induced IL-12 expression independently of IL-10 in macrophages, whereas in vivo administration of 11R-VIVIT was shown to significantly improve inflammatory lesions in an experimental model of colitis 39 . The leucine-rich repeat kinase 2 (LRRK2), identified as a major susceptibility gene for Crohn’s disease 40 41 , was reported to act as a negative regulator of NFAT1/c2-induced cytokine responses. LRRK2 modulates the cytoplasm retention of NFAT and the interaction between NFAT1 and the non coding RNA NFAT repressor (NRON) complex 42 in response to inducer of the innate immunity 43 . Furthermore, severe experimental colitis induced by dextran sulphate sodium (DSS) in LRRK2 deficient (Lrrk2 ^-/- ) mice was associated with enhanced nuclear localization of NFAT1. VIVIT was also shown to inhibit TNF-α induced expression in mouse bone marrow macrophages (BMMs) stimulated by the TLR4 ligand, LPS, and the TLR1/2 ligand, Pam3CSK4. In addition, LPS stimulation did not induce the nuclear translocation of NFAT1/c2 and NFAT2/c1, but in contrast, BMMs exhibited constitutive nuclear localization of NFAT4/c3 and NFAT3/c4, regardless of LPS stimulation. Moreover, VIVIT removed NFAT4 and NFAT3 from the nucleus and inhibited TLR-mediated activation of TNF 44 . Overall, these findings have highlighted regulatory roles of NFATs on different aspects of the immune response, and suggest that the NFATs may have distinct functions according to the cell type or pathogen considered.

Although the regulatory role of NFAT1 and other NFATs has been extensively studied in myeloid cells, only a few studies have analysed the expression and the role of NFAT/calcineurin signaling in neutrophils. Vega et al. 45 first reported the expression of NFAT2/c1 in human PMNs. Antigens, anti-IgE, and anti-FcϵR induced Ca²⁺ stimulation, which increases cellular calcineurin activity and the nuclear translocation of NFAT2/c1, while CsA and VIVIT abolished antigens- and anti-IgE-induced cyclooxygenase-2 (COX2) upregulation and prostaglandin E2 (PGE2) release 45 . Greenblatt et al. 46 also evidenced NFAT2/c1 and NFAT4/c3 expression in murine PMNs, and showed that mice with a conditional deletion of CnB in neutrophils, like CsA-treated mice, failed to control C. albicans infection without affecting the classical fungicidal activity, including ROS production and phagocytosis in response to C. albicans or zymosan stimulation. However, both CsA-treated neutrophils and CnB-deficient neutrophils exhibited impaired production of IL-10 after stimulation by zymosan and curdlan, which is the specific dectin-ligand with no TLR2 and TLR4 stimulating properties 46 . Altogether, these findings evidenced a novel, not yet fully characterized, NFAT-dependent and -independent candidacidal mechanism beyond dectin-1 that could account for the disseminated fungal infections observed in CsA-treated patients, independently of the effect of the immunosuppressive drugs on the adaptive immune response 46 . Recently, we also showed that knockdown of NFAT2/c1 mRNA expression by silencing mRNAs, similar to the inhibitory action of the 11R-VIVIT on NFATs, markedly inhibited the Nod1 mRNA expression without affecting Tlr2 and Tlr4 mRNA expressions in mouse macrophages activated by UPEC 47 . In vivo administration of CsA or FK506 (AV, ET, MB, CC, and CW, unpublished results), or 11R-VIVIT also markedly impaired the neutrophil bacterial phagocytic killing capacity of UPEC and increased the renal susceptibility to UPEC using an experimental murine model of ascending UTI 47 . These findings indicate that CsA may directly alter NOD1 expression and E. coli killing capacity by neutrophils. In line with these findings, a number of recent studies have provided evidence that NOD1 plays an important role in the regulation of neutrophil phagocytic function, which represents the main first line of defence against pathogenic bacteria. Figure  1 illustrates the participation of NFATc in the activation of the innate immune response in myeloid cells in response to various effectors. The following paragraphs will summarise the current knowledge of how NOD1 takes part in the regulation of the immune innate response to invasive pathogenic bacteria.

NOD1 (CARD4) belongs to the NLR family of multi-domain protein, including NOD2 (CARD15), and consists of an amino-terminal caspase activation and recruitment domain (CARD), a nucleotide-binding oligomerization domain (NBD), and a C-terminal leucine-rich repeat domain (LRR) 48 49 , that is also found in TLRs and that has been linked to resistance to infections 48 . The binding of MAMPs to the LRR domain results in the activation of signaling through homophilic CARD-CARD interactions 49 50 . NOD1 contains a single CARD domain, while NOD2 contains two domains 51 . NOD1 and NOD2 are two intracellular receptors that recognize bacterial peptidoglycan fragments. NOD1 recognizes γ-D-glutamyl-meso-diaminopimelic acid (meso-DAP), a degradation product of peptidoglycan (PGN) containing DAP 52 53 , which is present in most Gram-negative bacteria, such as Shigella flexneri, enteroinvasive and uropathogenic E. coli, Chlamydia, or Pseudomonas aeruginosa 47 54 55 56 57 , Helicobacter pylori 58 , and some Gram-positive bacteria 59 60 . In contrast, NOD2 recognizes muramyl dipeptide (MDP), a motif common to PGNs from all classes of bacteria 61 . NOD1 is ubiquitously expressed, while NOD2 is mainly found in macrophages, DCs, Paneth cells, and a variety of epithelial cells 13 . Mutations in the CARD15 gene encoding NOD2 have been shown to be associated with Crohn’s disease, a chronic inflammatory bowel disease mainly driven by T cells 62 63 .

Upon ligand recognition, the NBD domain of NOD1 or NOD2 oligomerizes and initiates the interaction of the CARD domain with RIPK2 (also called RIP2/RICK), which is a member of the CARD protein family 49 55 . RIPK2 is activated subsequently by proximity and promotes the formation of a signaling complex that contains the regulatory subunit of the IKK complex, NEMO 64 , leading to NF-κB activation (see Figure  2, left panel). NOD1 also induces the activation of c-Jun NH2-terminal kinase (JNK) pathway 55 65 and apoptosis 66 . NOD1 activating ligands were shown to enter cells through an endocytic process, most likely in a clathrin-dependent manner, and the cytosolic internalization of NOD1 ligands is pH-dependent 67 . NOD1 was also shown to be localized in the cytosol and plasma membrane of human intestinal cells, and imaging studies revealed that NOD1 is recruited to the site of entry of invasive S. flexneri 68 .

The rapid production of chemokines by immune cells and epithelial cells induces the rapid recruitment of polymorphonuclear neutrophils (PMNs) to the site of inflammation, which represent the first line of defence of the innate immune system against extracellular pathogens 69 . Masumoto et al. 70 first reported that the administration of the synthetic NOD1 ligand KF1B in WT mice induced the rapid production of CCL2/MCP1 and CXCL2/MIP-2 and the recruitment of intraperitoneal PMNs, but not lymphocytes and macrophages. This effect appeared to be NOD1-dependent since the recruitment of PMNs induced by the active NOD1-stimulatory compound KF1 was abolished in NOD1 deficient (Nod1 ^-/- ) mice. Dharancy et al. 71 also showed that upon liver injury induced by carbon tetrachloride (CCL4) intoxication, the in vitro migration capacity of PMNs induced by chemoattractants chemokines or formyl-methionyl-leucyl-phenylalanine (fMLP) was significantly reduced in Nod1 ^-/- PMNs compared to WT PMNs. These authors also showed that the number of infiltrating PMNs was lower in the liver from Nod1 ^-/- mice than WT mice subjected to ischemia-reperfusion injury. Conversely, FK565, another potent synthetic NOD1 agonist 72 , significantly stimulated the migration of PMNs 71 . Nod1 ^-/- mice were also shown to exhibit impaired production of CXCL1 and defective recruitment of neutrophils to the intestine after Clostridium difficile infection, suggesting that NOD1-mediated neutrophil recruitment regulates susceptibility towards C. difficile in the intestine 60 . We also showed using a murine model of ascending UTI that Nod1 ^-/- , but not Nod2 ^-/- mice, were more susceptible than WT mice to the retrograde inoculation of UPEC, and exhibited impaired recruitment of neutrophils in the UPEC-infected kidneys 47 .

The process governing PMN extravasation from blood vessels involves a complex multistep cascade that is orchestrated by a tightly coordinated sequence of adhesive interactions with vessel wall endothelial cells. The recruitment of PMNs involves a cascade of adhesive and migratory events, including the capture and selectin-mediated rolling of PMNs along the vessels, chemokine-induced activation of PMNs, and integrin-dependent adhesion and subsequent trans-endothelial migration 73 74 75 76 . Surface expression levels of the β2-integrin CD11b/CD18 decreased from about 50% in liver PMNs from CCL4-treated Nod1 ^-/- mice compared to that of their WT counterparts, and fMLP failed to activate CD11b expression in Nod1 ^-/- neutrophils 71 . In contrast, the synthetic agonist FK565 increased β2-integrin expression in neutrophils infiltrating injured liver after CCL4 intoxication. Renal bacterial loads were also significantly greater in the infected kidneys from Nod1 ^-/- mice than from their WT counterparts, 24 h after the transurethral inoculation of UPEC 47 . Like that occurring in Nod1 ^-/- livers subjected to ischemia 71 , the number of GR1^high+, CD11b^high+ neutrophils was also significantly less in kidneys from infected Nod1 ^-/- mice than in WT mice, 24 h after UPEC infection 47 .

The Weiser group demonstrated that NOD1 is involved in the phagocytic killing capacity of bone marrow-derived neutrophils 77 . This group showed, using a mouse model of airway bacterial co-infection with the Gram-positive pathogen Streptococcus pneumoniae and Gram-negative Haemophilus influenzae (Hi), that neutrophils from mice treated with the Gram-negative bacteria H. influenzae containing synthetic PGN fragments containing meso-DAP, activate cytoplasmic NOD1 and facilitate neutrophil opsonophagocytic killing of the Gram-positive Streptococcus pneumoniae 78 . Clarke et al. 79 then demonstrated that PGN can translocate from the gut into bone marrow cells, and that in vivo administration of a synthetic NOD1 ligand, but not the NOD2 ligand MDP, can restore neutrophil phagocytic functions after antibiotic-induced microbiota depletion 79 . These authors also showed that Nod1 ^-/- mice were more susceptible than WT mice to S. pneumoniae infection. Overall, these findings have provided clear evidence that PGN, through NOD1, can stimulate innate immunity in mouse neutrophils. These results are relevant to humans, since we found, ex vivo, in PMNs of human transplant patients a downregulation of NOD1 mRNA associated with reduced E. coli phagocytosis properties 47 .

The mechanism by which NOD1 stimulates bacterial phagocytosis has been first attributed to a priming effect of the immune system 79 , but recent studies evidenced close interplay between NOD1 and the small Rho GTPases 80 . Activated Rho GTPases Rac1 and Rac2, and Cdc42 81 82 , which lead to rapid actin rearrangements, play key roles in the activation of phagocytic processes 83 . Rac1 was shown to regulate PGN activation of the NF-κB signaling pathway through the recruitment of the p85 regulatory phosphoinositide 3-kinase (PI3K) subunit in macrophages 84 . Pathogenic bacteria also synthesize a number of virulent factors activating or mimicking small Rho GTPase proteins in host cells 85 . Most of the factors activating Rho GTPases have been identified in Gram-negative bacteria, such as the cytotoxic necrotizing Factor 1 (CNF1) expressed in UPEC strains, or the Salmonella outer protein E (SopE/SopE2) from S. Typhimurium 86 . These factors were shown to stimulate the innate immune response. For example, SopE/SopE2 and SopB from S. Typhimurium stimulate Rho GTPases leading to NF-κB and MAPKs activation 87 . The guanine nucleotide exchange factor H1 (GEF-H1) from S. flexneri, which interacts with NOD1, was shown to be required for the RIK2-dependent activation of NF-κB 88 . Boyer et al. 89 also showed that the E. coli cytotoxic necrotizing factor 1 (CNF1), the prototypal bacterial toxin activating host GTPases, activates Rac2, which then interacts with the innate immune adaptors IMD (the fly ortholog of RIP1/RIPK2), and RIP1 and RIPK2 to induce NF-κB activation and IL-8 expression in mammalian cells. Keestra et al. 90 using a mouse model of S. Typhimurium infection and transfected HEK293 cells, have shown that the activation of Rac1 and Cdc42 by bacterial delivery or SopE expression stimulate NOD1 signaling and downstream RIPK2-mediated stimulation of the NF-κB inflammatory response. These authors also showed that PGN detects NOD1 by sensing the activation of Rac1. These findings, which are summarised in the left panel from Figure  2, have provided the first direct demonstration that pathogen-induced NOD1 signaling requires small Rho GTPases.

The impact of immunosuppressive therapy, which increases susceptibility towards bacterial infection has been considered for a long time to be largely non specific. However, former studies have provided indirect evidence that calcineurin inhibitors may affect neutrophil functions 91 92 93 94 . The decreased renal susceptibility to UPEC following CsA treatment has been attributed to NFATc1-dependent inhibition of NOD1-mediated innate immune response 47 . Figure  2 summarises the main sites of inhibitory action of CsA and FK506, and VIVIT peptides on the Ca²⁺-dependent calcineurin/NFAT signaling leading to the activation of NFAT activated genes, such as IL-2. The inhibitory effects of these agents on NOD1 signaling pathway and subsequent activation of neutrophil functions are also shown.

CsA may differently affect immune receptors. CsA, which inhibits Nod1 mRNA expression in myeloid cells, also blunts Tlr4 mRNA expression without affecting Nod1 and Nod2 expression in renal tubule cells, suggesting that the combined decrease in TLR4 mRNA and protein expression in renal tubule cells and in NOD1 in myeloid cells caused by CsA should contribute to the observed decreased resistance to UPEC 47 . Immunosuppressant therapy increases the susceptibility towards bacterial and viral infection, which explains the incidence of infectious events in solid organ recipients. However, the concentrations of calcineurin inhibitors generally used in vitro are generally about 10-fold higher than those used in humans to prevent activation of lymphoid cells. The question arises as to whether or not the concentration of calcineurin inhibitors used during current immunosuppressive regimens are sufficient to fully suppress NFATc in myeloid cells. We reported that incubating murine myeloid cells with a concentration of 100 nM (~ 120 ng/ml) CsA, which is in the same range as the serum concentrations of CsA found in renal transplant recipients, markedly downregulated the expression of NOD1 (but not of TLR2 and TLR4). Clinical studies also evidenced a similar decrease in NOD1 mRNA and functional response in leukocytes from transplant recipients treated with CsA 47 . These findings suggest that low concentrations of CsA can be sufficient to impair NFAT activation and/or nuclear residence. By contrast to the observation that FK506 can induce reduced responsiveness to LPS in DCs and macrophages 95 , Tourneur et al. 47 did not evidence marked decrease in either LPS- or Pam3CSK4-induced IL-8 production in intact leukocytes from transplant recipients receiving CsA. Given that interplays between NODs and TLRs can be critical for the induction of protective immune responses 52 96 , it cannot be excluded that the possible induction of TLR4 tolerance together with decreased NOD1-mediated phagocytic functions caused by calcineurin inhibitors contribute to impaired resistance of transplant recipients to UPEC colonizing renal grafts. A better understanding of the mechanism of activation of small Rho GTPases and NOD1 by virulent effectors produced by pathogens should provide new insights in the mechanisms triggering bacterial phagocytosis.