Pacritinib abrogates the lupus phenotype in ABIN1[D485N] mice.

OBJECTIVE: The aim of the study was to investigate whether the IRAK1/JAK2/Flt3 inhibitor pacritinib prevents disease development in the lupus-prone ABIN1[D485N] knock-in mouse. METHODS: ABIN1[D485N] knock-in mice aged 8 weeks were fed for 10 weeks on a diet containing pacritinib. Body weight was monitored, and serum collected at the end to measure pacritinib, autoantibody and immunoglobulin levels. Splenic immune cell populations were analysed, and the kidney, liver and lungs examined for pathology. RESULTS: Pacritinib prevented multiple facets of the lupus phenotype in ABIN1[D485N] knock-in mice, including splenomegaly, expansion of splenic germinal centre B cells, follicular T helper cells, and neutrophils, elevated serum levels of double-stranded DNA antibodies and immunoglobulins, glomerular IgA and lung inflammation. CONCLUSIONS: Pacritinib may be useful for the treatment of multiorgan inflammation in patients with lupus.

TAK1 protein kinase activity is required for TLR signalling and cytokine production in myeloid cells.

A conditional knock-in mouse was generated in which the TAK1 catalytic subunit was largely replaced by the kinase-inactive TAK1[D175A] mutant in immune cells. The activation of p38α MAP kinase, c-Jun N-terminal kinases 1 and 2 (JNK1/2) and the canonical IKK complex induced by stimulation with several TLR-activating ligands was reduced in bone marrow-derived macrophages (BMDM) from TAK1[D175A] mice. TLR signalling in TAK1[D175A] BMDM was catalysed by the residual wild-type TAK1 in these cells because it was abolished by either of two structurally unrelated TAK1 inhibitors (NG25 and 5Z-7-oxozeaenol) whose off-target effects do not overlap. The secretion of inflammatory mediators and production of the mRNAs encoding these cytokines induced by TLR ligation was greatly reduced in peritoneal neutrophils or BMDM from TAK1[D175A] mice. The Pam3CSK4- or LPS-stimulated activation of MAP kinases and the canonical IKK complex, as well as cytokine secretion, was also abolished in TAK1 knock-out human THP1 monocytes or macrophages. The results establish that TAK1 protein kinase activity is required for TLR-dependent signalling and cytokine secretion in myeloid cells from mice. We discuss possible reasons why other investigators, studying myeloid mice with a conditional knock-out of TAK1 or a different conditional kinase-inactive knock-in of TAK1, reported TAK1 to be a negative regulator of LPS-signalling and cytokine production in mouse macrophages and neutrophils.

Why are the phenotypes of TRAF6 knock-in and TRAF6 knock-out mice so different?

The expression of TNF-Receptor Associated Factor 6 (TRAF6) is essential for many physiological processes. Here we studied the phenotype of TRAF6[L74H] knock-in mice which are devoid of TRAF6 E3 ligase activity in every cell of the body, but express normal levels of the TRAF6 protein. Remarkably, TRAF6[L74H] mice have none of the phenotypes seen in TRAF6 KO mice. Instead TRAF6[L74H] mice display an entirely different phenotype, exhibiting autoimmunity, and severe inflammation of the skin and modest inflammation of the liver and lungs. Similar to mice with a Treg-specific knockout of TRAF6, or mice devoid of TRAF6 in all T cells, the CD4+ and CD8+ T cells in the spleen and lymph nodes displayed an activated effector memory phenotype with CD44high/CD62Llow expression on the cell surface. In contrast, T cells from WT mice exhibited the CD44low/CD62Lhigh phenotype characteristic of naïve T cells. The onset of autoimmunity and autoinflammation in TRAF6[L74H] mice (two weeks) was much faster than in mice with a Treg-specific knockout of TRAF6 or lacking TRAF6 expression in all T cells (2-3 months) and we discuss whether this may be caused by secondary inflammation of other tissues. The distinct phenotypes of mice lacking TRAF6 expression in all cells appears to be explained by their inability to signal via TNF Receptor Superfamily members, which does not seem to be impaired significantly in TRAF6[L74H] mice.

Prevention and partial reversion of the lupus phenotype in ABIN1[D485N] mice by an IRAK4 inhibitor.

OBJECTIVE: We have reported previously that the IRAK4 inhibitor PF06426779 given to ubiquitin-binding-defective ABIN1[D485N] mice at 6 weeks of age prevents the major facets of lupus that develop 10 weeks later. The present study was undertaken to investigate whether PF06426779 could reverse the lupus phenotype when administered to 13-week-old ABIN1[D485N] mice that had already developed symptoms of lupus. METHODS: Splenomegaly, the number of splenic neutrophils, TFH and Germinal Centre B (GCB) cells, serum levels of immunoglobulins, the extent of kidney, liver and lung pathology, and glomerular IgA and IgM were measured after feeding 13-week-old ABIN1[D485N] and wild-type mice for another 10 weeks with R&M3 diet with and without PF06426779 (4 g/kg). RESULTS: Following drug treatment, spleen size and weight, splenic neutrophil numbers, and serum IgA and glomerular IgA levels of ABIN1[D485N] mice returned to those seen in wild-type mice. The rise in splenic TFH and GCB numbers, the increase in kidney and liver pathology, and the concentrations of serum IgG1, IgG2A and IgE between 13 and 23 weeks were suppressed. There was no reduction in the level of anti-self double-stranded DNA, anti-self nuclear antigens or IgM during the drug treatment. CONCLUSIONS: The results demonstrate the therapeutic potential of IRAK4 inhibitors for the treatment of lupus and raise the possibility of monitoring efficacy by measuring decreases in the serum levels of IgA. Our results support the view that there may be a closer connection between lupus and IgA nephropathy than realised previously.

HOIL-1-catalysed, ester-linked ubiquitylation restricts IL-18 signaling in cytotoxic T cells but promotes TLR signalling in macrophages.

The atypical E3 ligase HOIL-1 forms ester bonds between ubiquitin and serine/threonine residues in proteins, but the physiological roles of this unusual modification are unknown. We now report that IL-18 signalling leading to the production of interferon γ (IFNγ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) is enhanced in cytotoxic T cells from knock-in mice expressing the E3 ligase-inactive HOIL-1[C458S] mutant, demonstrating that the formation of HOIL-1-catalysed ester-linked ubiquitin bonds restricts the activation of this pathway. We show that the interaction of IRAK2 with TRAF6 is required for IL-18-stimulated IFN-γ and GM-CSF production, and that the increased production of these cytokines in cytotoxic T cells from HOIL-1[C458S] mice correlates with an increase in both the number and size of the Lys63/Met1-linked hybrid ubiquitin chains attached to IRAK2 in these cells. In contrast, the secretion of IL-12 and IL-6 and the formation of il-12 and il-6 mRNA induced in bone marrow-derived macrophages (BMDMs) by prolonged stimulation with TLR-activating ligands that signal via myddosomes, which also requires the interaction of IRAK2 with TRAF6, were not increased but modestly reduced in HOIL-1[C458S] BMDM. The decreased production of these cytokines correlated with reduced ubiquitylation of IRAK2. Our results establish that changes in HOIL-1-catalysed ester-linked ubiquitylation can promote or reduce cytokine production depending on the ligand, receptor and immune cell and may be explained by differences in the ubiquitylation of IRAK2.

Protection of the C. elegans germ cell genome depends on diverse DNA repair pathways during normal proliferation.

Maintaining genome integrity is particularly important in germ cells to ensure faithful transmission of genetic information across generations. Here we systematically describe germ cell mutagenesis in wild-type and 61 DNA repair mutants cultivated over multiple generations. ~44% of the DNA repair mutants analysed showed a >2-fold increased mutagenesis with a broad spectrum of mutational outcomes. Nucleotide excision repair deficiency led to higher base substitution rates, whereas polh-1(Polη) and rev-3(Polζ) translesion synthesis polymerase mutants resulted in 50-400 bp deletions. Signatures associated with defective homologous recombination fall into two classes: 1) brc-1/BRCA1 and rad-51/RAD51 paralog mutants showed increased mutations across all mutation classes, 2) mus-81/MUS81 and slx-1/SLX1 nuclease, and him-6/BLM, helq-1/HELQ or rtel-1/RTEL1 helicase mutants primarily accumulated structural variants. Repetitive and G-quadruplex sequence-containing loci were more frequently mutated in specific DNA repair backgrounds. Tandem duplications embedded in inverted repeats were observed in helq-1 helicase mutants, and a unique pattern of 'translocations' involving homeologous sequences occurred in rip-1 recombination mutants. atm-1/ATM checkpoint mutants harboured structural variants specifically enriched in subtelomeric regions. Interestingly, locally clustered mutagenesis was only observed for combined brc-1 and cep-1/p53 deficiency. Our study provides a global view of how different DNA repair pathways contribute to prevent germ cell mutagenesis.

p38 MAPK signalling regulates cytokine production in IL-33 stimulated Type 2 Innate Lymphoid cells.

Type 2 Innate lymphoid cells (ILC2s) are implicated in helminth infections and asthma where they play a role in the production of Th2-type cytokines. ILC2s express the IL-33 receptor and are a major cell type thought to mediate the effects of this cytokine in vivo. To study the signalling pathways that mediate IL-33 induced cytokine production, a culture system was set up to obtain pure populations of ILC2s from mice. Inhibitors of the p38α/ß and ERK1/2 MAPK pathways reduced the production of IL-5, IL-6, IL-9, IL-13 and GM-CSF by ILC2 in response to IL-33, with inhibition of p38 having the greatest effect. MK2 and 3 are kinases activated by p38α; MK2/3 inhibitors or knockout of MK2/3 in mice reduced the production of IL-6 and IL-13 (two cytokines implicated in asthma) but not IL-5, IL-9 or GM-CSF in response to IL-33. MK2/3 inhibition also suppressed IL-6 and IL-13 production by human ILC2s. MK2/3 were required for maximal S6 phosphorylation, suggesting an input from the p38α-MK2/3 pathway to mTOR1 activation in ILC2s. The mTORC1 inhibitor rapamycin also reduced IL-6 and IL-13 production, which would be consistent with a model in which MK2/3 regulate IL-6 and IL-13 via mTORC1 activation in ILC2s.

Distinct signals and immune cells drive liver pathology and glomerulonephritis in ABIN1[D485N] mice.

We report that TLR7, IL-6, and the adaptive immune system are essential for autoimmunity and glomerulonephritis but not for liver pathology in mice expressing the ubiquitin-binding-defective ABIN1[D485N] mutant. The blood and organs of ABIN1[D485N] mice have exceptionally high numbers of patrolling monocytes (pMo), which develop independently of IL-6 and the adaptive immune system. They are detectable in the blood months before autoimmunity and organ pathology are seen and may have diagnostic potential. The splenic pMo, inflammatory monocytes (iMo), and neutrophils of ABIN1[D485N] mice expressed high levels of mRNAs encoding proteins released during NETosis, which together with the high numbers of monocyte-derived dendritic cells (MoDCs) may drive the liver pathology in ABIN1[D485N] mice, and contribute to the pathology of other organs. The splenic iMo of ABIN1[D485N] mice displayed high expression of mRNAs encoding proteins controlling cell division and were actively dividing; this may underlie the increased pMo and MoDC numbers, which are derived from iMo. An orally active IRAK4 inhibitor suppressed all facets of the disease phenotype and prevented the increase in pMo numbers.

Loss of Functionally Redundant p38 Isoforms in T Cells Enhances Regulatory T Cell Induction.

The evolutionarily conserved protein kinase p38 mediates innate resistance to environmental stress and microbial infection. Four p38 isoforms exist in mammals and may have been co-opted for new roles in adaptive immunity. Murine T cells deficient in p38α, the ubiquitously expressed p38 isoform, showed no readily apparent cell-autonomous defects while expressing elevated amounts of another isoform, p38ß. Mice with T cells simultaneously lacking p38α and p38ß displayed lymphoid atrophy and elevated Foxp3+ regulatory T cell frequencies. Double deficiency of p38α and p38ß in naïve CD4+ T cells resulted in an attenuation of MAPK-activated protein kinase (MK)-dependent mTOR signaling after T cell receptor engagement, and enhanced their differentiation into regulatory T cells under appropriate inducing conditions. Pharmacological inhibition of the p38-MK-mTOR signaling module produced similar effects, revealing potential for therapeutic applications.