Background
Innate immunity is the early and relatively nonspecific response to invading pathogens, activated via the Toll-like and T-cell receptors, on antigen presenting cells and on T cells, respectively [
1,
2]. The intensity and duration of the immune response is under stringent regulation. Tyrosine phosphorylation is a central mechanism in the control of key signaling proteins involved in innate immunity. The role of protein tyrosine kinases (PTKs) has been widely studied but less is known on the protein tyrosine phosphatases (PTPs) responsible for immunoregulation [
3].
PTP action on immune response can be either positive or negative, promoting or inhibiting the immune system. SRC homology 2 (SH2)-containing tyrosine phosphatase-2 (SHP-2) has a controversial effect on lymphocyte signaling. Qu and colleagues demonstrated that SHP-2 is essential for erythroid and myeloid cell differentiation [
4], and a missense mutation in the ptpn11 gene (encoding for SHP-2 protein) is associated with various forms of leukemia [
5]. SHP-2 may also have an inhibitory role on the activation of T and B lymphocytes [
6]; SHP-2 can hamper the TRIF (TIR-domain-containing adapter-inducing interferon-β) adaptor protein-dependent TLR4 and TLR3 signal transduction with a consequent block of the pro-inflammatory cytokine production [
7]. Another negative regulator of hematopoietic cell development and function is SHP-1 (SRC homology 2 (SH2)-containing tyrosine phosphatase 1), that is mainly expressed in hematopoietic and lymphoid cells [
8]. Lymphocyte specific phosphatase, (LYP) and its mouse orthologue PEP (PTP enriched in proline, glutamic acid, serine, and threonine sequences) are predominantly expressed in leukocytes and act as potent negative regulators of the TCR signaling pathway [
9]. A specific missense mutation in the LYP encoding gene, ptpn22, has been associated in a highly reproducible manner with autoimmune disease, as type1 diabetes [
10] and rheumatoid arthritis [
11]. Another PTP involved in the immune processes is PTPMEG, a cytosolic phosphatase expressed in the thymus that is able to dephosphorylates TCRζ ITAMs
in vitro. Trapping mutant experiments show that PTPMEG inactivation leads to increased activation of the NF-kB pathway [
12]. However PTPMEG deletion
in vivo does not induce TCRζ ITAMs dephosphorylation, and PTPMEG-KO mice do not show obviously altered immune responses [
12].
The present study is focused on PTPH1 (also known as PTPN3), a cytosolic PTP that has been proposed to inhibit TCR signaling. PTPH1 overexpression in Jurkat T cells reduces indirectly the TCR-induced serine phosphorylation of Mek, Erk, Jnk and AP-1 leading to a decreased IL-2 gene activation [
13]. The indirect effect of PTPH1 could be mediated by the dephosphorylation of one or several signaling components upstream of Mek and Jnk, such as the TCR-associated protein tyrosine kinases (PTK) or their immediate targets. Further studies will be needed to identify the direct substrate for PTPH1. It has been also demonstrated that the FERM (band 4.1, ezrin, radixin, moesin) domain of PTPH1 is necessary for the inhibition of Mek, Erk, Jnk and AP-1 and also for localization of the phosphatase on the plasma membrane of Jurkat T cells [
14]. These studies corroborate the hypothesis of a possible role for PTPH1 as negative regulator in TCR signaling. Indeed, biochemical approaches and substrate trapping experiments identify PTPH1, together with SHP-1, as the phosphatases able to interact and to dephosphorylate TCRζ
in vitro[
15]. A comparatively recent
ex vivo study on PTPH1-KO primary T cells failed to show any significant role of this phosphatase in T cell development and activation, thus excluding a possible function for PTPH1 in the negative regulation of TCR signaling [
16]. This discrepancy between
in vitro and
ex vivo data has been explained by a possible redundancy effect of PTPMEG, that belongs to the same family protein of PTPH1. As already mentioned, PTPMEG is able to dephosphorylate the TCR ITAMs and to regulate NF-κB [
12]. Despite the similarity in protein structure between PTPMEG and PTPH1, no evidence can support the hypothesis of PTPH1 affecting NF-κB pathway. However, the double PTPH1-PTPMEG KO mouse line fails to show a T cell phenotype, indicating that PTPMEG does not compensate for the lack of PTPH1 action in primary T cells [
17].
In the present study, we examined the contribution of PTPH1 to the regulation of inflammatory responses in mice with a targeted deletion of PTPH1 gene expression. PTPH1-KO and WT mice were treated with two potent immunomodulatory molecules, carrageenan (CARR) and lipopolysaccharide (LPS). Nociceptive perception and cytokine expression and release have been investigated in these two models of local (carrageenan) and systemic (lipopolysaccharide) inflammation.
Discussion
PTPH1 has been proposed to act as a negative TCR regulator
in vitro, interacting and dephosphorylating the TCRζ chain [
13‐
15] but these results have not been confirmed by
ex vivo studies on primary PTPH1-KO T cells [
16,
17]. Therefore, we sought to ascertain whether PTPH1 could have an effect on immune system upon inflammatory challenge, thus in the complex
in vivo machinery. Two inflammatory mouse models were used to test the impact of PTPH1 deletion on the immune system: carrageenan- and LPS-induced inflammation.
Carrageenan λ is a sulfated polysaccharide derived from red seaweed that is able to activate the innate immune response. CARR interacts with TLR4 leading to increased Bcl10, to NFκB pathway activation and IL8 production [
25,
26]. CARR injection in the hind paw of the mouse is one of the most commonly used models of inflammation and inflammatory pain and it has a biphasic profile [
27]. Recent studies pointed out important roles for prostaglandins, nitric oxide and TNFα in the CARR-induced inflammatory response [
27‐
29]. In particular, it has been shown that TNFα is involved in both phases of mouse carrageenan-induced edema. Thus, TNFα has a strong relevance not only in inflammatory events, but also on nociceptive response and on neutrophil migration induced by carrageenan in mice [
29]. Soluble TNFα is processed from its pro-protein form by a specific sheddase, called TACE [
30,
31], that is also responsible for the processing of other cytokines and cytokine receptors [
32‐
35]. Interestingly, PTPH1 is known to inhibit TACE expression and activity
in vitro[
36]. We therefore analyzed cytokines plasma levels in carrageenan-treated WT and KO mice, but no variation was found between genotypes using this inflammatory agent (data not shown), in agreement with a previous CBA study on the rat carrageenan model [
37]. We conclude that local 2% carrageenan stimulation might not be sufficiently potent to unmask a phenotype in cytokine modulation in PTPH1-KO mice at plasma level, and that hind paw and muscle cytokine concentrations should be analyzed in both genotypes, to unravel PTPH1 role in local cytokine release.
As already mentioned, the CARR-induced model has a biphasic profile, that is characterized by an early development of edema, that peaks at 6 h and then again at 72 h [
27]. In the present study, carrageenan injection induced paw edema in both PTPH1-WT and KO mice, detectable already 1 hour after injection and persistent till 24 h (Figure
1), showing no differences in intensity between the genotypes. Furthermore, carrageenan challenge induced a marked neuthrophils migration to the site of injection 24 h after treatment (Figure
4) [
27]. Another hallmark of carrageenan stimulation is a long-lasting reduction in the threshold to nociceptive stimuli, that was evident in our model already 1 h after challenge and was sustained for up to 72 h [
27,
38,
39]. PTPH1-KO mice did not show any significant difference in neutrophils infiltration (Figure
4) or in pain behavior both at Von Frey's and Hargreaves' tests, compared to WT littermates (Figure
2a, 2b). These findings suggest that PTPH1 does not play a major role in the inflammatory-induced transmission and integration of the allodynic and painful stimuli. Comparatively, Catwalk gait analysis showed a trend of slightly earlier onset (5 h after injection) of spontaneous pain perception indicated as print area (Figure
3a) and duty cycle (Figure
3b) in PTPH1-KO mice, compared to matched WTs. Pilecka and colleagues recently showed that PTPH1 is expressed also in skeletal muscles [
40]. Despite no differences were detected in grip strength test between PTPH1-WT and KO mice in basal condition (data not shown), Catwalk data might also suggest a possible role of PTPH1 in muscle fatigue. Further tests should be performed on PTPH1-WT and KO mice upon challenge, in order to unravel the underlying molecular mechanisms.
Carrageenan and LPS challenges are frequently used in rodents as models to investigate innate immune response mechanisms [
41‐
43]. LPS is a major component of the outer membrane of Gram-negative bacteria and it is a critical player in the pathogenesis of septic shock [
44]. Like carrageenan, LPS binds to the MD2-TLR4 complex and activates both MyD88-dependent and independent (TRIF-dependent-TIR-domain-containing adapter-inducing interferon-β) pathways [
45‐
47]. The MyD88-dependent pathway results in the activation of TRAF6 (TNF Receptor Associated Factor 6) and in the immediate activation of NFκ B, MAPK and JNK pathways, leading to the early production of pro-inflammatory cytokines as TNFα, IL-1β, IL6 and MCP-1 [
48‐
50]. MyD88-independent pathway results in rapid activation of the interferon regulatory factors (IRF) 3 and 7 that induces the production of IFNβ and consequently IFNα, nitric oxide production and delayed NFκB activation [
48,
50], leading to late cytokine production. LPS challenge on PTPH1-KO and WT mice aimed to understand the possible role of this phosphatase in the inflammatory process and in particular in cytokine expression and release. Thus, CBA analysis was performed on the plasma of LPS- and vehicle-treated WT and KO mice (IL-10, IL-12p70, TNFα, IL-6, MCP-1, IFN-γ).
IL10 is an immunomodulatory cytokine, whose production is rapidly induced by monocyte/macrophages upon LPS challenge [
51]. IL10 treatment
in vitro is known to negatively regulate LPS responses [
51], in particular inhibiting the induction of pro-inflammatory cytokines, as TNFα, IL12 [
52,
53], IL-1α, and IL-6 [
54,
55]. Both PTPH1-WT and KO mice displayed significantly increased IL10 plasma level upon LPS challenge, but no significant reductions of IL-6, TNFα and thus MCP-1 plasma levels were detected in LPS-treated
vs vehicle-treated PTPH1-WT and KO mice. Interestingly, IL10 levels were reduced in LPS-treated KO mice plasma compared to WTs, at 30 and 60 minutes after challenge (Figure
6,
7); no increased MCP-1, TNFα, and IL6 plasma levels were detected in LPS-treated KO
vs WT mice at these time points. Indeed, IL-10 has not an exclusive pro-inflammatory action [
56] and it has been demonstrated in an LPS-model that IL-10 release increases as MCP-1, IL-6 and TNFα [
57]. Thus, our results on overall increased cytokines levels upon LPS treatment are in accordance with these studies. Comparatively, an overall decrease in cytokines release was recorded at 30 and 60 minutes post challenge in LPS-treated PTPH1-KO
vs WT mice (Figure
6,
7).
Several studies reported that IL10 up-regulates the expression of socs1 and socs3 genes, blocking the IFNs-induced JAK/STAT pathway [
51,
58] and stimulating the expression of PTP1B [
51]. It has recently been demonstrated that the production of IL10 by human Treg cells is enhanced by IL2 signaling via activation of STAT5 molecules [
59,
60]. PTPH1 is known to dephosphorylate STAT5b
in vitro[
61], and therefore the IL10 reduction in PTPH1-KO plasma after 30 minutes and 1 hour post LPS injection appears counterintuitive (Figure
6,
7). These data could indicate PTPH1 as a possible target of the early MyD88-dependent pathway, which acts on the overall pro- and anti-inflammatory cytokines production, but further investigations are needed to support this hypothesis and to identify PTPH1 substrates.
LPS-induced IL6 release was detectable in WT mice, LPS-treated
vs vehicle-treated group, as reported by several studies [
62,
63]. Increased IL6 plasma level of LPS
vs vehicle-treated mice were recorded also in KO mice, at the three time points investigated. It has been recently shown that JAK2 and STAT5 are required for LPS-induced IL-6 production [
63] and, as already mentioned, STAT5b is known as PTPH1 substrate [
61]. Thus, the trend in decreased IL6 plasma levels of KO
vs WT LPS treated mice 60 minutes after challenge could be due to a partial and temporally-limited inactivation of JAK/STAT pathway by PTPH1 deletion. Further biochemical analysis are necessary to confirm this hypothesis and to understand the possible PTPH1 role in this pathway.
It has been widely demonstrated that endotoxin injection leads to a rapid and dose-dependent TNFα expression and release in mice [
44,
64,
65]. The present study demonstrates that LPS injection induced an increased level of TNFα mRNA expression in LPS-treated
vs vehicle-treated mice in both PTPH1-WT and KO (Figure
5a, 5b, 5c), that is genotypically different only at 60 minutes after endotoxin injection (Figure
5b). At this time point, PTPH1-KO LPS-treated mice displayed lower TNFα expression compared to matched WTs, that was detectable also at the level of TNFα release in plasma (Figure
7). In particular, endotoxin injection led to a rapid and very significant increase of TNFα release in both PTPH1-WT and KO mice at the three time points investigated (Figure
7,
8). In accordance to mRNA level (Figure
5b), TNFα release was significantly lower in LPS-treated KO
vs matched WT mice 60 minutes post challenge (Figure
7), corroborating the hypothesis of a weaker inflammatory response of PTPH1-lacking mice.
Furthermore, increased MCP-1 plasma levels were recorded in both PTPH1-WT and KO LPS-treated
vs vehicle-treated mice (Figure
6,
7,
8). TNFα is known to promote ccl2 gene expression [
66,
67], activating NFκB-inducing kinases, including IκB kinases (IKK). IKKs promote NFκB heterodimers translocation into the nucleus, leading to the transcription of targeted genes [
66,
67]. Several studies show that both NFκB and MAPK pathways are required for ccl2 induction; specifically, transcription factor AP-1 contributes to TNFα-inducible expression of MCP-1 gene [
67‐
69]. This regulatory mechanism might explain the slight reduction of TNFα and MCP-1 gene expression and release in KO mice 1 hour post challenge (Table
1), when LPS induced the peak of TNFα plasma levels. Indeed, LPS challenge failed to induce a full response, as measured by TNFα expression and release in PTPH1-KO mice, leading to a lower stimulation of MCP-1 and possibly to a consequently reduced monocytes and macrophages recruitment 1 hour after injection. Cytokine levels in PTPH1-KO plasma were comparable to WT 3 hours after LPS injection (Figure
8), suggesting that PTPH1 activity was temporally limited at the peak of TNFα release, or that some compensatory mechanisms might have intervened later on, in the inflammatory process.
Table 1
Cytokine expression and release in LPS-treated mice- Summary
TNFα
| = | = | - | - | = | = |
MCP-1
| = | - | - (ns) | - | = | = |
IL6
| = | - (ns) | = | - (ns) | = | = |
IL12p70
| = | - | = | = | = | = |
IL 10
| nn | - | nn | - | nn | = |
Despite the fact that most pro-inflammatory cytokines are transcribed after NFκB activation [
68,
70‐
72], the overall control of production for several cytokines is more complex, and includes other pathways as JAK/STAT, MAPK [
73,
74], JNK [
75,
76] and also post-transcriptional and post-translational regulatory steps within these pathways, that could be cytokine-specific. PTPH1 could act at one or more steps of this composite regulatory process, by dephosphorylating key molecules, as indicated by the lack of direct correlation between cytokines expression and release, in particular for ccl2 (Table
1). Proteolytic conversion of pro-proteins into mature cytokines is a further level of control for cytokine production. TACE is involved in the ectodomain release of several cytokines, in particular of membrane-bound TNFα [
30,
77,
78]. As already mentioned, PTPH1 has been reported to be an inhibitor of TACE expression and activity
in vitro[
36], but the mechanism of this inhibition is still unclear.
Competing interests
The present work is part of CP's PhD program at the University of Eastern Piedmont, in close collaboration with MerckSerono International S.A VM and PFZ are former employees of MerckSerono International S.A RH and BG are employed by MerckSerono International S.A. which is involved in the discovery and the commercialization of therapeutics for the prevention and treatment of human diseases.
Authors' contributions
The study was devised by CP and VM and carried out by CP. DL performed the behavioral tests (Von Frey, Hargreaves and Catwalk) on PTPH1-WT and KO animals. VM, BG and RH have been deeply involved in the first editing of the manuscript and all the authors contributed to modifications in subsequent drafts. PFZ and RH have given the final approval of the version to be published. All the authors read and approved the final version of the manuscript.