Redox regulation of PTPN22 affects the severity of T cell dependent autoimmune inflammation

Chronic autoimmune diseases are associated with mutations in PTPN22, a modifier of T cell receptor signaling. As with all protein tyrosine phosphatases the activity of PTPN22 is redox regulated, but if or how such regulation can modulate inflammatory pathways in vivo is not known. To determine this, we created a mouse with a cysteine-to-serine mutation at position 129 in PTPN22 (C129S), a residue proposed to alter the redox regulatory properties of PTPN22 by forming a disulfide with the catalytic C227 residue. The C129S mutant mouse showed a stronger T cell-dependent inflammatory response and development of T cell dependent autoimmune arthritis due to enhanced TCR signaling and activation of T cells, an effect neutralized by a mutation in Ncf1, a component of the NOX2 complex. Activity assays with purified proteins suggest that the functional results can be explained by an increased sensitivity to oxidation of the C129S mutated PTPN22 protein. We also observed that the disulfide of native PTPN22 can be directly reduced by the thioredoxin system, while the C129S mutant lacking this disulfide was less amenable to reductive reactivation. In conclusion, we show that PTPN22 functionally interacts with Ncf1 and is regulated by oxidation via the non-catalytic C129 residue and oxidation-prone PTPN22 leads to increased severity in the development of T cell-dependent autoimmunity. Significance statement A hitherto unstudied aspect of PTPN22 biology is its regulation by cell redox states. Here we created a mouse model where PTPN22 was mutated to respond differentially to redox levels in vivo and found that PTPN22 function is regulated by reactive oxygen species and that redox regulation of PTPN22 impacts T-cell-dependent autoimmune inflammation.


Introduction
Complex autoimmune diseases affect 4-5% of the human population and large efforts have 61 been invested in finding the underlying genetic polymorphisms [1]. Though a major genetic 62 contribution comes from the major histocompatibility complex region, many other loci have 63 also been identified. Two important single nucleotide polymorphisms (SNPs) have emerged, 64 one located in PTPN22 [2], a cytoplasmic class I protein tyrosine phosphatase (PTP), and the balance between inhibitory oxidation of the catalytic Cys residue and its activation by 81 reduction, where the latter is typically maintained by the thioredoxin system. We have now 82 investigated the possibility that redox regulation of PTPN22 could modulate inflammatory 83 5 pathways in vivo. Interestingly, the PTPN22 catalytic cysteine (C227) has been suggested to 84 form a disulfide bond with another "back-door" cysteine (C129), possibly altering the 85 threshold for irreversible oxidation of C227 and thereby affecting the redox state of the active 86 site [10]. By creating a mouse with a cysteine-to-serine mutation at position 129 in PTPN22 87 (C129S) we could investigate the possible pathophysiological impact of its redox regulatory 88 properties in vivo. We found that mice with this amino acid replacement developed increased 89 T cell-dependent inflammatory responses due to enhanced T cell receptor signaling, which 90 was dependent on NOX2-produced ROS. This correlated well with findings of a lower overall 91 turnover, higher sensitivity to inhibitory oxidation, and a lower capacity of reductive 92 reactivation by the thioredoxin system of recombinant mutant PTPN22 C129S , as compared to 93 wild-type PTPN22. These results show that redox regulation of PTPN22 modulates 94 inflammation in vivo, with a lower resistance to oxidation of PTPN22 promoting aggravated 95 disease.

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Recombinant PTPN22 C129S has lower catalytic activity, higher sensitivity to inhibition by 98 oxidation and lower capacity for reductive reactivation by the thioredoxin system 99 To investigate the potential impact of a C129S replacement in the PTPN22 protein, we 100 recombinantly produced the catalytic domain of the corresponding human wild-type PTPN22 101 and PTPN22 C129S mutant proteins (the C129 and the catalytic C227 residues have the same 102 numbering in both mouse and human). The recombinant proteins were purified to >95% 103 purity as judged by SDS-PAGE and kinetic parameters were determined using p-NPP as 104 substrate; wild-type PTPN22 enzyme had a basal turnover of 19.6 min -1 with a Km of 4.57 mM 105 while PTPN22 C129S displayed a turnover of 11.9 min -1 and a Km of 8.02 mM under the same 106 6 conditions, showing that the C129S protein has retained PTP activity but with an overall lower 107 catalytic efficiency (Fig. 1A) which confirms Tsai et al.'s results [10]. 108 Next, we wanted to assess sensitivity of pre-reduced wild-type PTPN22 to inhibition by 109 oxidation. We found that addition of 50 µM H2O2 to the pure protein led to inhibition of 110 approximately half the activity after 20 min incubation (Fig. 1B). We also found that addition 111 of bicarbonate together with H2O2 noticeably potentiated the inactivation (Fig. 1C), similar to 112 the properties shown earlier for PTP1B that are likely due to formation of 113 peroxymonocarbonate that reacts more efficiently than H2O2 with the PTP enzyme [11]. When 114 comparing the H2O2-sensitivity of PTPN22 C129S with that of wild-type PTPN22, we found that 115 the mutant enzyme was clearly more sensitive to inhibition by H2O2 than wild-type PTPN22, 116 although it had a lower basal turnover (Fig. 1D). The same effect was also seen in the presence 117 of a functional thioredoxin system of thioredoxin reductase 1 (TrxR1) coupled with thioredoxin 118 (Trx1), but perhaps less so coupled with thioredoxin related protein of 14 kDa (TRP14) (Fig.   119 1E). It should be noted that also PTP1B displays different reactivities with Trx1 and TRP14 [12], 120 which may have physiological importance.

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True redox regulation of PTPN22 requires that the fully oxidized and thus inactivated enzyme 122 can be subsequently reactivated by reduction. Since the thioredoxin system has been 123 previously shown to reactivate phosphatases PTP1B and PTEN [11][13] [16], we next tested 124 this property. We found that H202-inactivated PTPN22 species could be re-activated in vitro 125 using either DTT as reductant, or the thioredoxin system composed of NADPH together with 126 TrxR1 and Trx1. Interestingly, wild-type PTPN22, but not the PTPN22 C129S mutant, could be 127 directly reactivated by TrxR1 together with NADPH, without inclusion of Trx1 ( Fig. 2A). 128 Subsequent experiments showed that the effect of TrxR1-dependent reactivation was 129 7 concentration dependent and that the oxidized forms of wild-type PTPN22 were clearly more 130 amenable to direct reactivation by TrxR1 than those of PTPN22 C129S (Fig. 2B). Since the only 131 difference between PTPN22 and PTPN22 C129S is the integrity of the non-catalytic C129 residue, 132 we reasoned that the direct reductive reactivation by TrxR1+NADPH of wild-type PTPN22, not 133 seen with the PTPN22 C129S mutant, might indicate that TrxR1 can directly reduce the disulfide 134 involving C129 that may be formed in the wild-type PTPN22 enzyme. To assess this possibility, 135 we subjected both forms of the enzyme to oxidative conditions, or to reduction by either DTT 136 or TrxR1, and then analyzed the enzymes on a non-reducing SDS-PAGE. Indeed, only PTPN22 137 but not PTPN22 C129S displayed a second faster migrating form of the protein upon oxidation 138 that disappeared upon reduction with DTT or TrxR1 (Fig. 2C). The effect was seen with several 139 concentrations of H2O2 and DTT treatment could always revert the double band of PTPN22 140 (Suppl. Fig. S1). We also found that the reactivation of wild-type PTPN22, with either DTT or 141 the thioredoxin system (TrxR1 alone, or coupled with either Trx1 or TRP14), was always more 142 efficient than that of the mutant PTPN22 C126S enzyme, also in cases where prior oxidation was 143 further potentiated by bicarbonate (Suppl. Fig. S2).

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These findings with recombinant human PTPN22 and PTPN22 C129S enzymes suggest that the 145 mutant is more amenable to oxidation and that the thioredoxin system is less efficient in 146 reactivating its oxidized form. The notion that TrxR1 directly can reduce the disulfide which is 147 not formed in the mutant enzyme, but which may be formed upon oxidation of wild-type 148 PTPN22, was interesting, as to our knowledge no PTP has earlier been shown to form a 149 disulfide species that is a direct substrate of TrxR1. Based upon these findings, we propose a 150 model for redox regulation of PTPN22 (Fig. 2D), which illustrates how PTPN22 C129S can be more 151 easily inactivated than the wild-type enzyme. Next, we wished to assess if mutant PTPN22 C129S 152 yields any phenotypic effects in mouse models of inflammation. To study redox-dependent regulation of PTPN22 in vivo and its possible downstream effects 155 on inflammation we generated a mouse with the PTPN22 C129S mutation (schematic in Fig. 3A).

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This disrupts the mechanism by which the catalytic C227 can be protected through formation 157 of a disulfide that can be reduced by TrxR1 (Fig. 2D), thus making PTPN22 C129S in the mice more 158 prone to inactivation by oxidation. The mice were backcrossed to C57Bl6/N mice together 159 with a major histocompatibility complex (MHC) region containing an Aq MHC class II allele, 160 making it susceptible to autoimmune arthritis [15], and also with the m1j mutation of Ncf1 161 [16], allowing interaction studies. 163 To study the effect of PTPN22 C129S on cell-mediated immunity we used the delayed-type 164 hypersensitivity (DTH) model, which is known to drive inflammation via IFNɣ producing type compared to PBS injection with the NCF1 mutants showing higher cell infiltration even in PBS-177 injected ears (Fig. 3F). Additionally, we observed higher CD4 expression in COL2-injected 178 PTPN22 ears as compared to wild-type with no difference in the NCF1 mutants (Fig.3G). To 179 further support the Th1 phenotype we performed qPCR analysis of the ears which showed 180 increased expression of CXCR3 in inflamed PTPN22 C129S ears (Fig. 3H).

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In the periphery, immunized PTPN22 C129S mice had increased cell numbers in the spleen as 182 compared to wild-type (Fig. 3I). Within the inguinal lymph nodes we observed a reduction in 183 CD4+ T cells which expressed higher levels of CD44, a marker for effector-memory T cells 184 (Fig.3J). This was not seen in circulating T cells within the blood, however there was a 185 significant reduction in FOXP3+ T cells in PTPN22 C129S mice 48 hours after initial immunization 186 (Fig. S3A).

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As we observed differences in T cell activation levels, we wanted to address the role of 188 PTPN22 C129S in antigen-specific T cells. To do this, we used the Vβ12-transgenic mouse (Vβ12-  Vβ12.PTPN22 C129S mice (Fig. 3L). 198 Together, these results indicate that the oxidation prone PTPN22 C129S mutant enhances T cell-199 mediated inflammation. Conversely, wild-type PTPN22 with a higher basal activity and more 200 resistance to oxidation counteracts these inflammatory processes.

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As PTPN22 is heavily associated with the development of autoimmunity we also sought to 203 explore the effects of PTPN22 C129S on arthritis development by using the glucose-6-phosphate-   221 To understand where the observed T cell activation phenotype in PTPN22 C129S mice originates 222 from, we first confirmed that PTPN22 expression was comparable between wild-type, 223 PTPN22 C129S , NCF1 m1J and PTPN22 C129S /NCF1 m1J mice, both via mRNA and protein analysis (

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As we consistently observed a differential T cell phenotype in mice with mutated PTPN22 C129S 240 in vivo, we studied T cell function in vitro. To assess signaling, we measured Ca 2+ -flux, one of reverted the phospho-PKC-θ levels in the mutant to be comparable with wild-type, followed 265 by a slower TCR-activation-dependent phosphorylation (Fig. 6C). Next, we investigated how 266 13 differential signaling was affecting the proteome signature using sorted CD4+ T cells from 267 wild-type and PTPN22 C129S mice. Mass spectrometric proteomics analysis of activated cells 268 revealed differential expression of multiple genes under both untreated and BSO-treated to be associated with increased PKC signaling (Fig. 6D).

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Thus, PTPN22 C129S that is prone to inactivation by oxidation with decreased catalytic activity 273 results in enhanced T cell signaling, which has broad signaling effects that can yield aggravated 274 inflammatory disease. should also be noted that the pure PTPN22 C129S migrated slightly slower than wild-type 327 PTPN22, despite the only difference between the two proteins being the single C129 residue.

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This also suggests an overall effect on protein characteristics by the C129S mutation, which 329 should be studied further. Based on our findings, we propose a model for the redox regulation 330 of PTPN22, with the disulfide bond that can be formed between C227 and C129 protecting the 331 catalytic C227 residue from overoxidation to sulphinic or sulphonic acid. It is also possible that 332 an alternative sulphenylamide motif can be made between the thiol group of the C227 with 333 the peptide bond amine, which can also be reduced by the thioredoxin system, similar to that 334 seen with PTP1B [13][14] even if that motif was not seen in the crystal structure of PTPN22 335 [10]. This model explains how PTPN22 C129S can still show activity and a certain protection 336 against oxidation and reactivation by the thioredoxin system, but simultaneously be more 337 susceptible to oxidation because the protective disulfide cannot be formed.

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In T cells, the PTPN22 C129S mutation led to enhanced T cell activation and proliferation. This 339 was not due to altered PTPN22 expression levels, but rather a consequence of downstream is a measure to combat inflammation, but where they originate from is a matter of discussion.  In summary, our results show how the activity of PTPN22, a gene where a risk variant is 383 associated with autoimmune diseases, can be regulated by ROS through its non-catalytic C129 384 residue, which is likely to have a major impact on its function in addition to that of merely 385 altered basal turnover (Fig.6D). It has been notoriously difficult to target PTPs in vivo with drug