Loss of PTPN22 leads to changes in cytokine secretion.

<p>(<b>A–C</b>) THP-1 cells, expressing non-targeting control or PTPN22 targeting shRNA, were treated for 24 h with 500 ng/ml MDP. The graphs show the secretion of (<b>A</b>) IL-6, (<b>B</b>) IL-8; or (<b>C</b>) TNF into the cell culture medium. (<b>D–F</b>) BMDC from WT or PTPN22 KO mice, were treated for 24 h with 500 ng/ml MDP and analysed for (<b>D</b>) IL-6, (<b>E</b>) IL-8; or (<b>F</b>) TNF secretion into the cell culture medium. (<b>G</b>) THP-1 cells were treated as in A–C; the graph shows levels of IFN-γ secretion. Values were shown in pg/ml and normalized to total protein content. Significant differences from the controls are denoted by asterisks (* = p<0.05, ** = p<0.01, *** = p<0.001); # = p<0.05, ## = p<0.01, ### = p<0.001 <i>vs.</i> MDP-treated control-transduced cells.</p

Autophagy is enhanced upon PTPN22 knockdown.

<p>THP-1 cells were treated for the indicated time with 500 ng/ml MDP. Western blots and results of densitometric analysis in the graphs below show levels of (<b>A+B</b>) LC3BI+II and β-actin; (<b>C</b>) ATG7 and β-actin; (<b>D</b>) ATG5 and β-actin; or (<b>E</b>) p62 and β-actin. Significant differences from the controls are denoted by asterisks (* = p<0.05, ** = p<0.01, *** = p<0.001); ## = p<0.01 <i>vs.</i> MDP-treated control-transduced cells.</p

Loss of PTPN22 enhances p38 and JNK MAPK phosphorylation.

<p><b>A–D:</b> THP-1 cells, expressing either non-targeting control shRNA or PTPN22 targeting shRNA, were treated for 30 min. with 500 ng/ml MDP. Representative Western blots and respective densitometric analysis show levels of (<b>A</b>) PTPN22 and β-actin; (<b>B</b>) phospho-p38 (Thr¹⁸⁰/Tyr¹⁸²) and total p38; (<b>C</b>) phospho-JNK (Thr¹⁸³/Tyr¹⁸⁵) and total JNK; (<b>D</b>) phospho-ERK (Tyr⁴²/Tyr⁴⁴) and total ERK. (<b>E+F</b>) BMDC from either wild type (WT) or PTPN22 knockout (PTPN22.KO) mice were stimulated for 30 min with 500 ng/ml MDP. Representative Western blots and densitometric analysis below show levels of (<b>E</b>) phospho-p38 (Thr¹⁸⁰/Tyr¹⁸²) and total p38; and (<b>F</b>) phospho-ERK (Tyr⁴²/Tyr⁴⁴) and total ERK. Asterisks denote significant differences from the respective control (n = 3 each, * = p<0.05, ** = p<0.01, *** = p<0.001); # = p<0.05, # = p<0.01, ### = p<0.001 <i>vs.</i> MDP-treatment of cells expressing control shRNA.</p

Loss of PTPN22 promotes the formation of autophagosomes.

<p>THP-1 cells, expressing either non-targeting control shRNA (<b>A–C</b>), or PTPN22 targeting shRNA (<b>D–F</b>) were left untreated, treated for 24 h with 500 ng/ml MDP or 100 nM rapamycine and stained for LC3B (red) and DAPI (blue). Representative pictures are shown for (<b>A</b>) non-treated cells expressing control shRNA, (<b>B</b>) MDP-treated cells expressing control shRNA, (<b>C</b>) Rapamycine-treated cells expressing control shRNA, (<b>D</b>) non-treated cells expressing PTPN22 shRNA, (<b>E</b>) MDP-treated cells expressing PTPN22 shRNA; and (<b>F</b>) Rapamycine-treated cells expressing PTPN22 shRNA. Scale bar: 10 µm.</p

Changed mRNA expression in BMDC lacking PTPN22.

<p>BMDC from either wild-type (WT) or PTPN22 knockout (PTPN22.KO) mice, were treated 24 h with 500 ng/ml MDP. The graphs depict levels of (<b>A</b>) IL-6; (<b>B</b>) TNF; (<b>C</b>) NOD2; (<b>D</b>) ICAM-1; or (<b>E</b>) IFN-γ mRNA normalized to β-actin and relative to non-treated WT BMDC. Asterisks denote significant differences from the respective control (* = p<0.05, ** = p<0.01, *** = p<0.001); # = p<0.05, ## = p<0.001, <i>vs.</i> MDP-treatment of WT BMDC.</p

Knockdown of PTPN22 promotes canonical but not non-canonical NF-κB activation.

<p>(<b>A+B</b>) THP-1 cells, expressing non-targeting control or PTPN22 specific shRNA, were treated for 30 min with 500 ng/ml MDP. Western blots and densitometric analysis below show levels of (<b>A</b>) phospho-IκB-α (Ser³²) and total IκB-α; (<b>B</b>) phospho-NF-κB p65 (Ser⁵³⁶) and total NK-κB p65. (<b>C</b>) BMDC from either wild type (WT) or PTPN22 knockout (PTPN22.KO) mice were treated for 30 min with 500 ng/ml MDP. Representative Western blots and densitometric analysis below show levels of phospho-NF-κB p65 (Ser⁵³⁶) and total NK-κB p65. (<b>D</b>) THP-1 cells were treated as in A+B, Western blots and densitometric analysis show levels of phospho-NF-κB p105 (Ser⁹³³) and total NF-κB p105. (<b>E</b>) BMDC were treated as in (C); Western blot and densitometric analysis show levels of phospho-NF-κB p105 (Ser⁹³³) and total NF-κB p105. (<b>F</b>) THP-1 cells were treated as in A+B, Western blots and densitometric analysis show levels of phospho-NF-κB p50 (Ser⁹³³) and total NF-κB p50. Asterisks denote significant differences from the respective control (* = p<0.05, ** = p<0.01, *** = p<0.001); # = p<0.05, ### = p<0.001 <i>vs.</i> MDP-treatment of cells expressing control shRNA.</p

Changed mRNA expression upon knockdown of PTPN22.

<p>THP-1 cells, expressing either control or PTPN22 specific shRNA, were treated 24 h with 500 ng/ml MDP. The graphs depict levels of (<b>A</b>) IL-6; (<b>B</b>) IL-8; (<b>C</b>) NOD2; (<b>D</b>) ICAM-1; (<b>E</b>) T-bet; or (<b>F</b>) IFN-γ mRNA normalized to β-actin and relative to non-treated control-shRNA expressing cells. Asterisks denote significant differences from the respective control (*** = p<0.001); # = p<0.05, # = p<0.001, ### = p<0.001 <i>vs.</i> MDP-treatment of cells expressing control shRNA.</p

Site of opportunistic infection.

<p>Site of opportunistic infection.</p

Control group.

<p>Control group.</p

Details of opportunistic infections during lymphopenia.

<p>Details of opportunistic infections during lymphopenia.</p