4.1. PTPN22—Allowing Efficient NLRP3 Activation via Its Dephosphorylation
PTPN22 is exclusively expressed in innate and adaptive immune cells [
74,
75] and controls immune responses in multiple ways. Initial studies identified PTPN22 as a potent negative regulator of T-cell receptor (TCR) signaling [
76] via dephosphorylation of key signaling molecules downstream of the T-cell receptor (TCR), such as TCRζ, Lck and ζ—chain-associated protein (ZAP)70 [
76,
77]. By limiting the activation of naïve and effector T-cells [
78,
79,
80,
81], PTPN22 maintains immunological tolerance and homeostasis via suppression of autoreactive T-cell responses. In genome-wide associations studies (GWAS), the single nucleotide polymorphism (SNP) rs2476601 within the PTPN22 locus was identified to increase the risk for autoimmune disorders such as type 1 diabetes (T1D) [
82], rheumatoid arthritis (RA) [
83], systemic lupus erythematosus [
84,
85] and Graves’ disease [
86]. Paradoxically, the very same variant has been associated with a decreased risk to develop Crohn’s disease (CD) [
87]. Presence of SNP rs2476601 results in the substitution of arginine 620 (arginine 619 in mice) with a tryptophan residue resulting in an altered function protein product referred to as R620W variant. In a study to determine PTPN22 function in myeloid cells, Wang et al. described that PTPN22 controls host defense and type 1 interferon (IFNs) immune responses [
88]. PTPN22 directly binds and activates TRAF3, thus promoting toll-like-receptor (TLR) signaling [
88]. However, the disease-associated variant of PTPN22 failed to induce TRAF3 ubiquitination and PTPN22-variant expressing cells failed to mount efficient type I IFN responses [
88]. In mouse models of experimental arthritis and DSS colitis, PTPN22 deficiency resulted in enhanced susceptibility and pronounced progression of disease, indicating that PTPN22 is required to protect from systemic and gastrointestinal inflammation [
88]. Together, these data strengthened the conclusions from previous findings, suggesting that PTPN22 is a multifactorial regulator of innate and adaptive immune response [
78,
79,
80,
81,
88].
While exploring the function of PTPN22 in macrophages and monocytes, we identified NLRP3 as a direct interaction partner of PTPN22 [
55]. PTPN22 knockdown in THP-1 cells and loss of PTPN22 in primary murine macrophages resulted in reduced NLRP3-dependent IL-1β secretion upon stimulation with well-known NLRP3 activators. On the contrary, the autoimmunity-associated PTPN22 variant enhanced IL-1β secretion [
18]. Immunoblotting revealed that PTPN22 directly interacted with NLRP3 and this interaction was enhanced upon inflammasome activation. Subsequently we demonstrated that PTPN22 promotes inflammasome activation via direct dephosphorylation of Tyr861 in NLRP3 (
Figure 1). This process depended on the presence of the inflammasome adaptor molecule ASC, which was required for stabilization of NLRP3-PTPN22 protein-protein interaction [
55]. Interestingly, the R619W variant of PTPN22 was found to constitutively interact with NLRP3, consequently decreasing inhibitory NLRP3 phosphorylation and thereby promote its activation [
55]. A follow-up study revealed that PTPN22 regulates NLRP3 activation in an autophagy-dependent manner [
64]. NLRP3 Tyr681 phosphorylation resulted in its sequestration into phagophores, diminishing inflammasome activity. NLRP3 dephosphorylation by PTPN22 protected NLRP3 from the recruitment into phagophores and thereby prevented its degradation. Notably, the autoimmunity-associated PTPN22 variant was more effective in NLRP3 dephosphorylation [
64].
Mice expressing the murine R619W ortholog of the human R629W PTPN22 variant are protected in acute DSS colitis [
88]. Having shown that PTPN22 expression levels are decreased in intestinal biopsies of IBD patients [
89], we revealed additional immune-modulatory roles of PTPN22 in controlling autophagy and cytokine secretion [
90]. Both loss of NLRP3 as well as PTPN22 resulted in aggravation of acute DSS colitis, while presence of the R619W variant protected the animals from developing intestinal inflammation [
88]. Studying the molecular mechanisms, we found reduced expression levels of mature caspase-1, IL-1β and IL-18 in the lamina propria of inflamed PTPN22 KO mice but not in the epithelial cell compartment. In WT mice, DSS treatment resulted in reduced NLRP3 phosphorylation in epithelial and lamina propria cells, whereas adverse effects were observed in the lamina propria of PTPN22 KO mice [
55]. Collectively, our data showed that tyrosine phosphorylation of NLRP3 suppressed its activation, and that this is directly regulated by PTPN22 [
55]. The R619W PTPN22 variant, however, resulted in a gain of function with regards to NLRP3 activation [
55], which resulted from enhanced substrate affinity, as well as increased phosphatase activity [
55,
91,
92].
The NLRP3 inflammasome has been associated with the pathogenesis of different auto-inflammatory diseases such as Crohn’s disease, Alzheimer’s disease, diabetes, and atherosclerosis [
24,
93,
94]. The autoimmunity-associated PTPN22 variant promotes NLRP3 activity, caspase-1 activation, and secretion of IL-1β and IL18, which might—at least partially—explain the pro-inflammatory character of this PTPN22 variant in T1D, RA and SLE development. In the model of acute MSU peritonitis, we demonstrated that IL-1β-mediated neutrophil infiltration was reduced in PTPN22 KO mice whereas presence of the R619W variant aggravated peritonitis [
55]. Nevertheless, while promoting disease in other organs, the PTPN22 variant had protective effects in the setting of intestinal inflammation. Although surprising at a first glance, colonic NLRP3 expression is known to promote wound healing, host defense against invading bacteria [
94,
95,
96], and promotes gut homeostasis and barrier integrity [
97,
98,
99]. In a mouse model of acute DSS colitis, NLRP3 deficiency resulted in more severe barrier defects, erosion of the epithelium, infiltration of immune cells and alteration of the microbiome [
98,
100], probably due to the fact that myeloid-cell derived IL-1β in the intestine might promote wound healing and tissue repair [
101].
4.2. PTPN2—Negative Regulation of Inflammasomes via Inhibition of ASC Phosphorylation
PTPN2 is closely related to PTPN22 and exerts prominent anti-inflammatory functions in the body. PTPN2 is expressed in all cell types and controls several pro-inflammatory mediators, including signal transducer and activator of transcription (STAT) molecules [
102,
103], c-Jun N-terminal kinase (JNK) and ERK [
104,
105,
106], and thereby controls signaling cascades downstream of a multitude of cytokine and growth factor receptors [
106,
107,
108]. PTPN2 is also responsible for the dephosphorylation of molecules downstream of the T and B cell receptor, such as Lck and Fyn [
109]. Given these diverse anti-inflammatory effects, it is not surprising that genetic deletion of
PTPN2 results in generalized and uncontrolled inflammatory responses, and full-body
PTPN2 knockout mice succumb to severe systemic inflammation 3–5 weeks after birth [
110]. Variants in the gene locus encoding
PTPN2 are associated with several inflammatory disorders, including IBD [
111], RA [
112], and diabetes [
113], highlighting the important anti-inflammatory function of PTPN2. Notably, these disorders have all been associated with elevated levels of IL-1β.
With respect to inflammasome activation, PTPN2 has a dual regulatory effect: via interference with inflammatory signaling cascades [
104,
105], PTPN2 negatively regulates expression of inflammasome components, such as IL-1β and IL-18 while also directly affecting inflammasome assembly and subsequent release of active IL-1β and IL-18 [
114]. Loss of PTPN2 results in elevated ASC tyrosine phosphorylation and subsequent ASC-multimer formation, as well as enhanced caspase-1 processing/activation and subsequent maturation and release of IL-1β and IL-18 [
114]. However, although ASC activity is regulated via tyrosine phosphorylation, we found that PTPN2-mediated control of inflammasome activity was not mediated via direct interaction of PTPN2 with ASC, but that the effect of PTPN2 on ASC activity is mediated indirectly via modulation of JNK activity [
114] (
Figure 1). Although it has been shown JNK and Syk activity is essential for mediating ASC phosphorylation and ASC-speck formation [
70,
71], neither JNK nor Syk directly phosphorylate ASC, but they both promote the activity of the tyrosine phosphatase Pyk2, which is the kinase ultimately responsible for ASC tyrosine phosphorylation and subsequent promotion of its activation [
72]. This shows that there are several instances upstream of ASC phosphorylation that might serve as possible points of interference to prevent inadequate and/or overshooting inflammasome activation.
Regulation of ASC-mediated inflammasome activation by PTPN2 is critical for reducing inflammatory responses to several well-described inflammasome activators, such as flagellin, particulate molecules (i.e., MSU crystals or asbestos) or bacterial toxins [
114]. Defective PTPN2-mediated inflammasome control had profound effects in the setting of intestinal inflammation, where loss of PTPN2 in myeloid cells resulted in highly elevated IL-1β levels that promoted the perpetuation of inflammatory reactions, resulting in increased IL-1β-dependent intestinal inflammation [
114]. Nevertheless, in the context of malignancies, such as colorectal carcinoma, increased inflammasome activity upon loss of PTPN2 had a benign effect via promotion of anti-tumor immune responses, which allowed more efficient tumor cell eradication [
114]. Furthermore, increased inflammasome activation upon loss of PTPN2 might also contribute to the increased disease risk for RA and diabetes observed in
PTPN2-variant carriers [
113,
115].
4.3. SHP-2—Interference with Mitochondrial-Damage-Associated NLRP3 Activation
NLRP3 responds to several cell damage-inducing compounds, and as mentioned above, it is still not fully understood how NLRP3 reacts to a wide range of stimuli with apparently different molecular and structural nature. However, many of these compounds promote oxidative stress and mitochondrial damage either directly or indirectly [
21,
116], which brings Src homology 2 (SH2) domain-containing tyrosine phosphatase-2 (SHP2, encoded by the gene
PTPN11) into the picture of inflammasome-modulating tyrosine phosphatases. Like PTPN2, SHP2 is expressed in all tissues of the body, and it is involved in numerous signaling cascades, including growth factor and cytokine receptor induced signaling [
117]. Although SHP2 is a phosphatase, it has signaling-promoting functions in several of these pathways. Specifically, SHP2 is required for efficient and sustained Ras-Raf-ERK activation downstream of growth hormone receptors, such as the epidermal growth factor (EGF) receptor, human EGF receptor 2 (her2), and Insulin-like growth factor (IGF) receptor [
118,
119,
120]. The signaling promoting function of SHP2 seems to depend on its phosphorylation status and requires the two SH-domains present in SHP2, but its phosphatase function might be dispensable [
121]. In T cells, SHP2 promotes checkpoint inhibitor molecule programmed cell death (PD)-1 induced signaling [
121,
122], while simultaneously dephosphorylating and inactivating ZAP70, a kinase downstream of the T cell receptor that mediates T cell receptor-induced T cell activation and proliferation. In this way SHP2 dampens T cell receptor-induced expansion of effector T cells, and allows for the immune-evasion of cancer cells [
122]. Thus, SHP2 not only promotes potentially pro-oncogenic signaling pathways, but also inhibits anti-tumor T cell functions. Given its cell growth and survival promoting functions, and its involvement in dampening tumor-eradicating immune responses, SHP2 has been doubt “a proto-oncogenic tyrosine phosphatase” [
119], and has emerged as a target for cancer treatment. In addition to those signaling promoting effects downstream of growth hormone receptors, SHP2 negatively regulates TLR3- and TLR4-mediated JNK, ERK and p38 activation via interfering with the TLR-adaptor molecule TRIF, and dampens TLR3 and TLR4-induced type-1 interferon and TNFα production [
123]. In this way, SHP2 might also interfere with inflammasome component accumulation downstream of TLR ligation.
With regards to regulating inflammasome activation directly, Guo et al. first noticed that SHP2-deletion promoted NLRP3-mediated inflammasome activation [
124], although this was not due to a direct interaction with and/or dephosphorylation of NLRP3. Instead, SHP2-deletion promoted oxidative stress and resulted in increased levels of mitochondrial damage, which was responsible for the elevated NLRP3-mediated inflammasome activity observed in SHP2-deficient cells [
124]. The authors demonstrated that under normal conditions, NLRP3 activation resulted in the recruitment of SHP2 to mitochondria where it bound to and dephosphorylated ANT-1 [
124]. Under physiological conditions, ANT-1 is involved in exchanging cytosolic adenosine di-phosphate with mitochondrial adenosine tri-phosphate, thus ensuring energy supply to the cell and preventing oxidative stress [
125]. However, under situations of cellular stress, phosphorylated ANT-1 can promote disruption of the inner mitochondrial membrane, which leads to mitochondrial damage [
126]. In this way, ANT-1 not only contributes to apoptosis initiation [
127], but also potentiates NLRP3 inflammasome assembly (
Figure 1) [
21]. SHP-2 counteracts ANT-1-induced mitochondrial damage via direct dephosphorylation of ANT-1 [
124]. In this way, SHP2 restores mitochondrial membrane integrity/prevents mitochondrial damage and thereby prevents sustained NLRP3 activation [
124]. Notably, the study by Guo et al. reported that NLRP3 activation preceded mitochondrial damage and SHP2 mitochondrial translocation [
124], thus mitochondrial damage potentiated NLRP3 activity, but seemed not to be responsible for its initial activation.
While macrophage-specific ablation of SHP2 promoted susceptibility to inflammasome-driven, experimental peritonitis [
124], it also prevented high fat diet-induced obesity, an effect dependent on the release of IL-18 from macrophages [
128]. This observation highlights the finding that depending on the context, inflammasomes can have devastating effects, but in certain settings they are protective and might have disease-preventing effects.
4.4. PTP-S2—Promoting Caspase-1 Overexpression via Regulation of p53
PTP-S2 is a nuclear tyrosine phosphatase that binds non-specifically to DNA within the cell nucleus. PTP-S2 is upregulated during mitosis and promotes progression through the G1 to the S phase [
129,
130], indicating a functional role in cell proliferation. Furthermore, overexpression of PTP-S2 induced cell death in cells that express the tumor suppressor gene p53, but not in p53 negative cells [
131], indicating a negative feed-back loop to prevent excessive proliferation. p53 mediates anti-proliferative and anti-tumor effects via induction of cell cycle arrest and it induces apoptosis via upregulation of several cell-death associated molecules [
132]. In resting cells, the half-life of p53 is relatively short, resulting in low cytosolic levels of p53 [
132]. Upon DNA-damage or hyperproliferative signals, however, posttranscriptional modifications promote p53 stability [
132], allowing for the expression of cell-death-inducing target genes. While p53 induces several genes that mediate immune-silent apoptosis [
132,
133], it also promotes the expression of the inflammasome effector molecule caspase-1 [
134]. Via p53 dephosphorylation, PTP-S2 promoted stability, accumulation, and transcriptional activity of p53, which resulted in very high cytosolic levels of caspase-1 [
135]. Despite the normally very tight regulation of caspase-1 activity, excessive cytosolic caspase-1 accumulation mediates its aberrant activation, and finally inflammatory cell death [
135]. Although this regulatory mechanism has been described in the artificial setting of PTP-S2 overexpression, it demonstrates that regulation of the transcription of inflammasome components can be modulated via the activity of tyrosine phosphatases, introducing an additional mode of action how protein tyrosine phosphatases regulate inflammasome function.