B cell autophagy mediates TLR7-dependent autoimmunity and inflammation

<p>Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease, defined by loss of B cell self-tolerance that results in production of antinuclear antibodies (ANA) and chronic inflammation. While the initiating events in lupus development are not well defined, overexpression of the RNA-recognizing toll-like receptor (TLR)7 has been linked to SLE in humans and mice. We postulated that autophagy plays an essential role in TLR7 activation of B cells for the induction of SLE by delivering RNA ligands to the endosomes, where this innate immune receptor resides. To test this hypothesis, we compared SLE development in <i>Tlr7</i> transgenic (Tg) mice with or without B cell-specific ablation of autophagy (<i>Cd19-Cre Atg5</i>^<i>f/f</i>). We observed that in the absence of B cell autophagy the 2 hallmarks of SLE, ANA and inflammation, were eliminated, thus curing these mice of lupus. This was also evident in the significantly extended survival of the autophagy-deficient mice compared to <i>Tlr7.1</i> Tg mice. Furthermore, glomerulonephritis was ameliorated, and the serum levels of inflammatory cytokines in the knockout (KO) mice were indistinguishable from those of control mice. These data provide direct evidence that B cells require TLR7-dependent priming through an autophagy-dependent mechanism before autoimmunity is induced, thereafter involving many cell types. Surprisingly, hyper-IgM production persisted in <i>Tlr7.1</i> Tg mice in the absence of autophagy, likely involving a different activation pathway than the production of autoantibodies. Furthermore, these mice still presented with anemia, but responded with a striking increase in extramedullary hematopoiesis (EMH), possibly due to the absence of pro-inflammatory cytokines.</p

The Impact of Genetic Susceptibility to Systemic Lupus Erythematosus on Placental Malaria in Mice

<div><p>Severe malaria, including cerebral malaria (CM) and placental malaria (PM), have been recognized to have many of the features of uncontrolled inflammation. We recently showed that in mice genetic susceptibility to the lethal inflammatory autoimmune disease, systemic lupus erythematosus (SLE), conferred resistance to CM. Protection appeared to be mediated by immune mechanisms that allowed SLE-prone mice, prior to the onset of overt SLE symptoms, to better control their inflammatory response to <i>Plasmodium</i> infection. Here we extend these findings to ask does SLE susceptibility have 1) a cost to reproductive fitness and/or 2) an effect on PM in mice? The rates of conception for WT and SLE susceptible (SLE^s) mice were similar as were the number and viability of fetuses in pregnant WT and SLE^s mice indicating that SLE susceptibility does not have a reproductive cost. We found that <i>Plasmodium chabaudi</i> AS (<i>Pc)</i> infection disrupted early stages of pregnancy before the placenta was completely formed resulting in massive decidual necrosis 8 days after conception. <i>Pc</i>-infected pregnant SLE^s mice had significantly more fetuses (∼1.8 fold) but SLE did not significantly affect fetal viability in infected animals. This was despite the fact that <i>Pc</i>-infected pregnant SLE^s mice had more severe symptoms of malaria as compared to <i>Pc</i>-infected pregnant WT mice. Thus, although SLE susceptibility was not protective in PM in mice it also did not have a negative impact on reproductive fitness.</p></div

DataSheet_1_Plasmodium curtails autoimmune nephritis via lasting bone marrow alterations, independent of hemozoin accumulation.pdf

The host response against infection with Plasmodium commonly raises self-reactivity as a side effect, and antibody deposition in kidney has been cited as a possible cause of kidney injury during severe malaria. In contrast, animal models show that infection with the parasite confers long-term protection from lethal lupus nephritis initiated by autoantibody deposition in kidney. We have limited knowledge of the factors that make parasite infection more likely to induce kidney damage in humans, or the mechanisms underlying protection from autoimmune nephritis in animal models. Our experiments with the autoimmune-prone FcγR2B[KO] mice have shown that a prior infection with P. yoelii 17XNL protects from end-stage nephritis for a year, even when overall autoreactivity and systemic inflammation are maintained at high levels. In this report we evaluate post-infection alterations, such as hemozoin accumulation and compensatory changes in immune cells, and their potential role in the kidney-specific protective effect by Plasmodium. We ruled out the role of pigment accumulation with the use of a hemozoin-restricted P. berghei ANKA parasite, which induced a self-resolved infection that protected from autoimmune nephritis with the same mechanism as parasitic infections that accumulated normal levels of hemozoin. In contrast, adoptive transfer experiments revealed that bone marrow cells were altered by the infection and could transmit the kidney protective effect to a new host. While changes in the frequency of bone marrow cell populations after infection were variable and unique to a particular parasite strain, we detected a sustained bias in cytokine/chemokine expression that suggested lower fibrotic potential and higher Th1 bias likely affecting multiple cell populations. Sustained changes in bone marrow cell activation profile could have repercussions in immune responses long after the infection was cleared.</p

Increased CD40 Expression Enhances Early STING-Mediated Type I Interferon Response and Host Survival in a Rodent Malaria Model

<div><p>Both type I interferon (IFN-I) and CD40 play a significant role in various infectious diseases, including malaria and autoimmune disorders. CD40 is mostly known to function in adaptive immunity, but previous observations of elevated CD40 levels early after malaria infection of mice led us to investigate its roles in innate IFN-I responses and disease control. Using a <i>Plasmodium yoelii nigeriensis</i> N67 and C57BL/6 mouse model, we showed that infected CD40^-/- mice had reduced STING and serum IFN-β levels day-2 post infection, higher day-4 parasitemia, and earlier deaths. CD40 could greatly enhance STING-stimulated luciferase signals driven by the IFN-β promoter <i>in vitro</i>, which was mediated by increased STING protein levels. The ability of CD40 to influence STING expression was confirmed in CD40^-/- mice after malaria infection. Substitutions at CD40 TRAF binding domains significantly decreased the IFN-β signals and STING protein level, which was likely mediated by changes in STING ubiquitination and degradation. Increased levels of CD40, STING, and ISRE driven luciferase signal in RAW Lucia were observed after phagocytosis of N67-infected red blood cells (iRBCs), stimulation with parasite DNA/RNA, or with selected TLR ligands [LPS, poly(I:C), and Pam3CSK4]. The results suggest stimulation of CD40 expression by parasite materials through TLR signaling pathways, which was further confirmed in bone marrow derived dendritic cells/macrophages (BMDCs/BMDMs) and splenic DCs from CD40^-/-, TLR3^-/- TLR4^-/-, TRIF^-/-, and MyD88^-/- mice after iRBC stimulation or parasite infection. Our data connect several signaling pathways consisting of phagocytosis of iRBCs, recognition of parasite DNA/RNA (possibly GPI) by TLRs, elevated levels of CD40 and STING proteins, increased IFN-I production, and longer host survival time. This study reveals previously unrecognized CD40 function in innate IFN-I responses and protective pathways in infections with malaria strains that induce a strong IFN-I response, which may provide important information for better understanding and management of malaria.</p></div

CD40 modulates STING ubiquitination and degradation through interaction with TRAFs.

<p>(<b>A</b>) Co-immunoprecipitation (co-IP) of CD40 and STING. Co-IP of CD40 and STING 24 h after transfecting genes encoding CD40 and STING. Anti-HA antibody was used to pull-down protein complex in 293T cells transfected with DDK-tagged CD40, and proteins were detected using anti-HA (STING) or anti-DDK (CD40). No direct interaction of CD40 and STING was detected. (<b>B</b>) Co-IP using anti-Myc to pull-down CD40 and anti-HA to pull-down STING and immunoblot (IB) detection of TRAF molecules after co-transfection with TRAF2, TRAF3, or TRAF6, respectively. (<b>C</b> and <b>D</b>) The same experiments as in (<b>B</b>), but pulling-down using anti-Myc (CD40) and detection using anti-HA (STING) (<b>C</b>), or vice versa (<b>D</b>). (<b>E</b>) Effects of over expression of TRAF2, TRAF3, or TRAF6 on CD40 and STING protein levels 24 h after co-transfection of plasmids with indicated genes. (<b>F</b>) The same experiments as (<b>E</b>) but 48 h after co-transfection. (<b>G</b>) Detection of protein ubiquitination after co-transfection of plasmids encoding HA-tagged wild type or mutant ubiquitins with specific lysine (K) sites and with or without those encoding CD40 and STING. Anti-Myc was used to detect STING. (<b>H</b>) The same experiments as in (<b>G</b>), except Myc tagged STING was pulled-down before detection of HA-tagged ubiquitins. All the assays were performed in 293T cells (2 × 10⁵). IB, immunoblotting; IP, immunoprecipitation (pulled-down).</p

CD40 enhances IFN-β expression through STING/MAVS mediated pathways.

<p>(<b>A</b>) IFN-β level, measured as luciferase signals driven by IFN-β promoter, in 293T cells after transfection of a pCMV plasmid containing the gene encoding CD40 and stimulated with the indicated agents. Luciferase activities were measured 18 h after transfection of 200 ng plasmid each. (<b>B</b>) Co-transfection of 293T cells with plasmids containing the <i>cd40</i> and the indicated genes. IFN-β level, measured as luciferase signals driven by IFN-β promoter in 293T cells, were measured as in (<b>A)</b>. (<b>C-E</b>) Dose-response assays as done in <b>(B)</b>. (<b>F</b>) IFN-β protein levels in culture supernatants of RAW Lucia cells measured using ELISA after stimulation with cGAMP or interferon stimulatory DNA (ISD) for 6 h; both are STING ligands. All data are means+s.d. from three experiments; <i>t</i>-test, *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001.</p

Stimulation of CD40 and STING expression and ISRE driven luciferase activities by TLR ligands and infected red blood cells (iRBCs).

<p>(<b>A</b>) RAW Lucia cells (2 × 10⁵) were stimulated with different ligands for indicated times, and protein levels of CD40 and STING were detected using an antibody against C-terminus of CD40 and anti-STING, respectively. Pam is Pam3CSK4. (<b>B</b>) Plots of protein band intensities of CD40 and STING and ISRE-luciferase signals 1, 8, and 24 h after infected red blood cell (iRBC) stimulation. All data for ISRE-luciferase signals are averaged values from three experiments. (<b>C</b>) The same plots as in (<b>B</b>) after infected poly(I:C) stimulation. (<b>D</b>) The same plots as in (<b>B</b>) except stimulation with LPS. (<b>E</b>) Western blot detection of CD40 protein levels in 293T cells (2 × 10⁵) after TLR ligand stimulations for 12 h. (<b>F</b>) Expression of CD40 in 293T cells (2 × 10⁵) transfected with various TLRs for 18 h and with or without ligand stimulations for another 12 h before harvesting for proteins and Western blot analysis. The three TLR3 and TLR4 experiments, respectively, were conducted using plasmids containing the genes from different sources.</p

Mutations in CD40 TRAF binding domains affect STING mediated IFN-β response.

<p>(<b>A</b>) Diagram of CD40 showing various amino acid substitutions in the TRAF binding domains. For example, mT6 (Q238A, E239A) stands for mutations in TRAF 6 binding domain with amino acid substitution at Q238A and E239A. Mbx2 is the box2 at the C-terminal of CD40 as described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005930#ppat.1005930.ref017" target="_blank">17</a>]. (<b>B</b>) Luciferase signals driven by IFN-β promoter from 293T cells (2 × 10⁵) transfected with plasmids carrying wild type (WT) or different <i>Cd40</i> mutant genes were measured 24 h after lipofectamine transfection. Mutations in one or more TRAF binding domains/box are as indicated. (<b>C</b>) The same experiments as in (<b>B</b>) with co-transfection of <i>Sting</i> gene. (<b>D)</b> Luciferase signals derived by NFκB promoter from 293T cells transfected with plasmids carrying WT or mutations in TRAF23 or TRAF6 domains of CD40. (<b>E</b>) The same experiments as (<b>D)</b> but co-transfected with <i>Sting</i> gene. (<b>F</b>) STING protein expression levels after co-transfection of 293T cells (2 × 10⁵) with WT <i>Cd40</i> or <i>Cd40</i> with mutations in the TRAF binding domains for 24 h, and cell lysates were detected on Western blot. (<b>G</b>) Quantitative measurements of protein levels in (<b>F)</b>. For (<b>B-E</b>), data are means+s.d. from three experiments; <i>t</i>-test, *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001.</p

Stimulation of CD40 expression and ISRE driven luciferase signals by infected red blood cells (iRBCs), parasite DNA/RNA, or parasite GPI.

<p>(<b>A</b>) Flow cytometry counts of RAW Lucia cells containing N67 iRBCs 1 h and 8 h post incubation. The top two panels are RBCs, and the bottom panels are iRBCs. SSC, side-scattered light; CF-SE, signals of carboxyfluorescein succinimidyl ester (CF-SE) labeled RBCs or iRBCs (<b>B</b>) CD40 protein expression in RAW Lucia cells after incubation with RBCs or iRBCs. The antibody was against an epitope at C-terminus of the CD40 protein. Anti-β-actin was used as protein loading control. (<b>C</b>) The same as (<b>B</b>) but in DC2.4 cells. (<b>D</b>) ISRE promoter driven luciferase signals in RAW Lucia cells after ingestion with RBCs or iRBCs. (<b>E</b>) CD40 protein expression in RAW Lucia cells stimulated with parasite genomic DNA or RNA with or without the presence of lipofectamine. (<b>F</b>) Luciferase activities driven by ISRE promoter after stimulation with parasite DNA or RNA as in (<b>E</b>). (<b>G</b>) CD40 protein expression in RAW Lucia cells stimulated with parasite DNA or RNA with or without DNase or RNase treatment, respectively. EDTA and/or lipofectamine (Lipo) were used as buffer controls. (<b>H</b>) Luciferase activities driven by ISRE promoter after stimulation with parasite DNA or RNA with or without DNase or RNase treatment, respectively, as in (<b>G</b>). (<b>I</b>) CD40 protein expression in RAW Lucia cells stimulated with LPS, Pam3CSK4, and <i>Plasmodium falciparum</i> GPI (glycophosphatidylinositol) dissolved in ethanol (marked with EH) or H₂O. (<b>J</b>) Protein band intensities in (<b>I</b>) relative to non-stimulated in ethanol (NoneEH = 1). (<b>K</b>) Luciferase activities driven by ISRE promoter in RAW Lucia cells stimulated with LPS, Pam3CSK4, and <i>P</i>. <i>falciparum</i> GPI as in (<b>I</b>). For (<b>D</b>), (<b>F</b>), (<b>H</b>), and (<b>K</b>), data are means+s.d. from three experiments; <i>t</i>-test, *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001. For all the experiments, 5 × 10⁵ cells were used.</p

Effects of TLR signaling on CD40 and STING expressions in stimulated BMDCs and DCs from KO mice.

<p>(<b>A</b>-<b>D</b>) CD40, STING, and IFN-β expressions in BMDCs from WT and TLR3^-/- mice after stimulation. The cells were stimulated with the indicated agents for 24 h before harvesting proteins for Western blot. (<b>A</b>), Western blots; (<b>B</b> and <b>C</b>), quantitative signals of protein bands of CD40 and STING, respectively; (<b>D</b>) secreted IFN-β measured using ELISA. (<b>E-H</b>) the same as (<b>A</b>-<b>D)</b>, except in TLR4^-/- mice. (<b>I</b> and <b>J</b>) CD40 and STING expressions in purified splenic DCs from various KO mice after incubation with iRBCs for 24 h. Proteins from two mice each stimulated with iRBCs were separated on Western blot (<b>I</b>) and averaged signals were plotted in bar graphs (<b>J</b>). (<b>K</b> and <b>L</b>) CD40 and STING expressions in purified splenic DCs 4 days after infection with N67. Proteins were detected on Western blot (<b>K</b>) and scanned signals (means+s.d.) from three mice each were plotted (<b>L)</b>. <i>t</i>-test, *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001. For all the experiments, 5 × 10⁵ cells were used.</p