Mitochondrial reactive oxygen species suppress humoral immune response through reduction of CD19 expression in B cells in mice

Reactive oxygen species (ROS) are implicated in the modulation of diverse processes including immune responses. To evaluate the effects of metabolic ROS produced by mitochondria on B‐cell function and development, we created transgenic (Tg) mice expressing a phosphorylation‐defective mutant of succinate dehydrogenase A in B cells (bSDHA^Y215F). Splenic B cells in male, but not female, bSDHA^Y215F mice produced three times more ROS than those in the control mice, and had decreased production of IgM, IgG₁, and IgG₃, and affinity maturation of IgG₁ against T‐cell‐dependent antigens. Following immunization, the male bSDHA^Y215F mice further displayed suppressed germinal center (GC) formation, and proliferation of GC B cells. Signaling analysis revealed defects in the intrinsic BCR responses, such as activation of Lyn, Btk, and PLCγ2, thus resulting in reduced intracellular Ca²⁺ mobilization. Notably, the expression levels of B‐cell co‐receptor CD19 and its interaction with Lyn after BCR ligation were significantly reduced in B cells from male bSDHA^Y215F mice. These results suggest that mitochondrial ROS suppress humoral immune responses through reduction of CD19 expression and resultant BCR signaling in B cells. Therefore, B‐cell immunity may be more labile to oxidative stress in male mice than in female mice.     

Gp91<Superscript>phox</Superscript> (NOX2) in classically activated microglia exacerbates traumatic brain injury

Background  

We hypothesized that gp91^phox (NOX2), a subunit of NADPH oxidase, generates superoxide anion (O₂ ^-) and has a major causative role in traumatic brain injury (TBI). To evaluate the functional role of gp91^phox and reactive oxygen species (ROS) on TBI, we carried out controlled cortical impact in gp91^phox knockout mice (gp91^phox-/-). We also used a microglial cell line to determine the activated cell phenotype that contributes to gp91^phox generation.     

NADPH oxidase inhibition improves neurological outcomes in surgically-induced brain injury

Neurosurgical procedures can result in brain injury by various means including direct trauma, hemorrhage, retractor stretch, and electrocautery. This surgically-induced brain injury (SBI) can cause post-operative complications such as brain edema. By creating a mouse model of SBI, we tested whether NADPH oxidase, an important reactive oxygen species producing enzyme, is involved in SBI using transgenic mice lacking gp91^phox subunit of NADPH oxidase (gp91^phox KO) and apocynin, a specific inhibitor of NADPH oxidase. Neurological function and brain edema were evaluated at 24 h post-SBI in gp91^phox KO and wild-type littermates grouped into SBI and sham-surgery groups. Alternatively, mice were grouped into vehicle- and apocynin-treated (5 mg/kg, i.p. 30 min before SBI) groups. Oxidative stress indicated by lipid peroxidation (LPO) was measured at 3 and 24 h post-SBI. The gp91^phox KO mice, but not the apocynin-treated mice showed significantly improved neurological scores. Brain edema was observed in both gp91^phox KO and wild-type groups after SBI; however, there was no significant difference between these two groups. Brain edema was also not affected by apocynin-pretreatment. LPO levels were significantly higher in SBI group in both gp91^phox KO and wild-type groups as compared to sham group. A trend, although without statistical significance, was noted towards attenuation of LPO in the gp91^phox KO animals as compared to wild-type group. LPO levels were significantly attenuated at 3 h post-SBI by apocynin-pretreatment but not at 24 h post-SBI. These results suggest that chronic and acute inhibition of NADPH oxidase activity does not reduce brain edema after SBI. Long-term inhibition of NADPH oxidase, however improves neurological functions after SBI.     

B and CD4+ T‐cell expression of TLR2 is critical for optimal induction of a T‐cell‐dependent humoral immune response to intact Streptococcus pneumoniae

TLR2^?/? mice immunized with Streptococcus pneumoniae (Pn) elicit normal IgM, but defective CD4⁺ T‐cell‐dependent type 1 IgG isotype production, associated with a largely intact innate immune response. We studied the T‐cell‐dependent phosphorylcholine (PC)‐specific IgG3 versus the T‐cell‐independent IgM response to Pn to determine whether TLR2 signals directly via the adaptive immune system. Pn‐activated TLR2^?/? BMDC have only a modest defect in cytokine secretion, undergo normal maturation, and when transferred into naïve WT mice elicit a normal IgM and IgG3 anti‐PC response, relative to WT BMDC. Pn synergizes with BCR and TCR signaling for DNA synthesis in purified WT B and CD4⁺T cells, respectively, but is defective in cells lacking TLR2. Pn primes TLR2^?/? mice for a normal CD4⁺ T‐cell IFN‐γ recall response. Notably, TLR2^?/? B cells transferred into RAG‐2^?/? mice with WT CD4⁺T cells, or TLR2^?/? CD4⁺T cells transferred into athymic nude mice, each elicit a defective IgG3, in contrast to normal IgM, anti‐PC response relative to WT cells. These data are the first to demonstrate a major role for B‐cell and CD4⁺ T‐cell expression of TLR2 for eliciting an anti‐bacterial humoral immune response.     

Concomitant type I IFN receptor‐triggering of T cells and of DC is required to promote maximal modified vaccinia virus Ankara‐induced T‐cell expansion

Virus‐induced expansion of CD8⁺ T cells may be promoted by type I IFN receptor (IFNAR)‐triggering of T cells, depending on the pathogen tested. We studied modified vaccinia virus Ankara (MVA), a promising vaccine vector candidate, which was derived from conventional vaccinia virus (VACV) by more than 570 consecutive in vitro passages. In adoptive transfer experiments, we verified that VACV expressing the gp33 epitope of lymphocytic choriomeningitis virus (VACV_gp33) induced largely IFNAR‐independent expansion of gp33‐specific T cells. On the contrary, MVA_gp33‐induced T‐cell expansion was IFNAR dependent. Interestingly, under the latter conditions, T‐cell activation was IFNAR independent, whereas T‐cell apoptosis was enhanced in the absence of IFNAR. To address whether MVA‐induced T‐cell expansion was solely affected by IFNAR‐triggering of T cells, expansion of endogenous T cells was studied in conditional mice with a T‐cell‐ or DC‐specific IFNAR deletion. Interestingly, both mouse strains showed moderately reduced T‐cell expansion, whereas mice with a combined T‐cell‐ and DC‐specific IFNAR ablation showed massively reduced T‐cell expansion similar to that of IFNAR^?/? mice. These results are compatible with the model that IFN‐inducing viruses such as MVA confer virus‐specific CD8⁺ T‐cell expansion by concomitant IFNAR‐triggering of DC and of T cells.     

Constitutive Akt1 signals attenuate B‐cell receptor signaling and proliferation,but enhance B‐cell migration and effector function

Ligation of the BCR induces a complex signaling network that involves activation of Akt, a family of serine/threonine protein kinases associated with B‐cell development, proliferation, and tumor formation. Here, we analyzed the effect of enhanced Akt1 signals on B‐cell maturation and function. Unexpectedly, we found that peripheral B cells overexpressing Akt1 were less responsive to BCR stimuli. This correlated with a decrease in Ca²⁺‐mobilization and proliferation, in an impaired activation of Erk1/2 and mammalian target of rapamycin (mTOR) kinases and poor mobilization of NFATc1 and NF‐κB/p65 factors. In contrast, activation of STAT5 and migration of B cells toward the chemokine SDF1α was found to be enhanced. Akt1 Tg mice showed an altered maturation of peritoneal and splenic B1 B cells and an enhanced production of IgG1 and IgG3 upon immunization with the T‐cell independent Ag TNP‐Ficoll. Furthermore, mice homo‐zygous for Tg Akt1 showed a severe block in the maturation of B‐cell precursors in BM and a strong enrichment of plasma cells in spleen. Altogether, our data reveal that enhanced Akt1 signals modify BCR signaling strength and, thereby, B‐cell development and effector function.     

Cardiolipin activates antigen‐presenting cells via TLR2‐PI3K‐PKN1‐AKT/p38‐NF‐kB signaling to prime antigen‐specific naïve T cells in mice

Mitochondrial defects and antimitochondrial cardiolipin (CL) antibodies are frequently detected in autoimmune disease patients. CL from dysregulated mitochondria activates various pattern recognition receptors, such as NLRP3. However, the mechanism by which mitochondrial CL activates APCs as a damage‐associated molecular pattern to prime antigen‐specific naïve T cells, which is crucial for T‐cell‐dependent anticardiolipin IgG antibody production in autoimmune diseases is unelucidated. Here, we show that CL increases the expression of costimulatory molecules in CD11c⁺ APCs both in vitro and in vivo. CL activates CD11c⁺ APCs via TLR2‐PI3K‐PKN1‐AKT/p38MAPK‐NF‐κB signaling. CD11c⁺ APCs that have been activated by CL are sufficient to prime H‐Y peptide‐specific naïve CD4⁺ T cells and OVA‐specific naïve CD8⁺ T cells. TLR2 is necessary for anti‐CL IgG antibody responses in vivo. Intraperitoneal injection of CL does not activate CD11c⁺ APCs in CD14 KO mice to the same extent as in wild‐type mice. CL binds to CD14 (Kd = 7 × 10^?7 M). CD14, but not MD2, plays a role in NF‐kB activation by CL, suggesting that CD14⁺ macrophages contribute to recognizing CL. In summary, CL activates signaling pathways in CD11c⁺ APCs through a mechanism similar to gram (+) bacteria and plays a crucial role in priming antigen‐specific naïve T cells.     

Epratuzumab modulates B‐cell signaling without affecting B‐cell numbers or B‐cell functions in a mouse model with humanized CD22

Treatment of systemic lupus erythematosus patients with epratuzumab (Emab), a humanized monoclonal antibody targeting CD22, leads to moderately reduced B‐cell numbers but does not completely deplete B cells. Emab appears to induce immunomodulation of B cells, but the exact mode of action has not been defined. In the present study, we aimed to understand the effects of Emab on B cells using a humanized mouse model (Huki CD22), in which the B cells express human instead of murine CD22. Emab administration to Huki CD22 mice results in rapid and long‐lasting CD22 internalization. There was no influence on B‐cell turnover, but B‐cell apoptosis ex vivo was increased. Emab administration to Huki CD22 mice had no effect on B‐cell numbers in several lymphatic organs, nor in blood. In vitro exposure of B cells from Huki CD22 mice to Emab resulted in decreased B‐cell receptor (BCR) induced Ca²⁺ mobilization, whereas B‐cell proliferation after Toll‐like receptor (TLR) stimulation was not affected. In addition, IL‐10 production was slightly increased after TLR and anti‐CD40 stimulation, whereas IL‐6 production was unchanged. In conclusion, Emab appears to inhibit BCR signaling in a CD22‐dependent fashion without strong influence on B‐cell development and B‐cell populations.     

Swiprosin‐1/EFhd2 limits germinal center responses and humoral type 2 immunity

Activated B cells are selected for in germinal centers by regulation of their apoptosis. The Ca²⁺‐binding cytoskeletal adaptor protein Swiprosin‐1/EFhd2 (EFhd2) can promote apoptosis in activated B cells. We therefore hypothesized that EFhd2 might limit humoral immunity by repressing both the germinal center reaction and the expected enhancement of immune responses in the absence of EFhd2. Here, we established EFhd2^?/? mice on a C57BL/6 background, which revealed normal B‐ and T‐cell development, basal Ab levels, and T‐cell independent type 1, and T‐cell independent type 2 responses. However, T cell‐dependent immunization with sheep red blood cells and infection with the helminth Nippostrongylus brasiliensis (N.b) increased production of antibodies of multiple isotypes, as well as germinal center formation in EFhd2^?/? mice. In addition, serum IgE levels and numbers of IgE⁺ plasma cells were strongly increased in EFhd2^?/? mice, both after primary as well as after secondary N.b infection. Finally, mixed bone marrow chimeras unraveled an EFhd2‐dependent B cell‐intrinsic contribution to increased IgE plasma cell numbers in N.b‐infected mice. Hence, we established a role for EFhd2 as a negative regulator of germinal center‐dependent humoral type 2 immunity, with implications for the generation of IgE.     

Prenatal Diagnosis of X‐Linked Chronic Granulomatous Disease by Percutaneous Umbilical Blood Sampling

In this study, we investigated how to prenatally diagnose X‐linked chronic granulomatous disease (X‐CGD) effectively and accurately. Percutaneous umbilical blood sampling was conducted in the 22nd week of pregnancy. NADPH oxidase activity and gp91^phox protein expression of neutrophils were analysed using flow cytometry. Direct sequencing was used to detect CYBB gene mutations. Umbilical blood was obtained from seven foetuses whose mothers were X‐CGD carriers. Six foetuses, whose mothers needed prenatal diagnosis because of other diseases, were used as control. The neutrophils in all 13 foetuses showed lower hydrogen peroxide generation (stimulation index < 100) and gp91^phox protein expression. Among the seven foetuses whose mothers were X‐CGD carriers, four foetuses (Family 2, 4, 5 and 7) had CYBB gene mutations and showed very low hydrogen peroxide generation (stimulation index < 10) and no gp91^phox expression. The other three foetuses (Family 1, 3 and 6) had no CYBB gene mutations and showed stimulation index of 20–50 and partial or normal gp91^phox protein expression (no difference with controls). Two of the three mothers (Family 1 and 3) have delivered healthy infants with normal hydrogen peroxide generation and expression of gp91^phox in neutrophils. Combined with direct sequencing, dihydrorhodamine oxidation analysis and gp91^phox protein detection is an effective and accurate method for prenatal screening for X‐CGD.     

JNK2 modulates the CD1d‐dependent and ‐independent activation of iNKT cells

Invariant natural killer T (iNKT) cells play critical roles in autoimmune, anti‐tumor, and anti‐microbial immune responses, and are activated by glycolipids presented by the MHC class I‐like molecule, CD1d. How the activation of signaling pathways impacts antigen (Ag)‐dependent iNKT cell activation is not well‐known. In the current study, we found that the MAPK JNK2 not only negatively regulates CD1d‐mediated Ag presentation in APCs, but also contributes to CD1d‐independent iNKT cell activation. A deficiency in the JNK2 (but not JNK1) isoform enhanced Ag presentation by CD1d. Using a vaccinia virus (VV) infection model known to cause a loss in iNKT cells in a CD1d‐independent, but IL‐12‐dependent manner, we found the virus‐induced loss of iNKT cells in JNK2 KO mice was substantially lower than that observed in JNK1 KO or wild‐type (WT) mice. Importantly, compared to WT mice, JNK2 KO mouse iNKT cells were found to express less surface IL‐12 receptors. As with a VV infection, an IL‐12 injection also resulted in a smaller decrease in JNK2 KO iNKT cells as compared to WT mice. Overall, our work strongly suggests JNK2 is a negative regulator of CD1d‐mediated Ag presentation and contributes to IL‐12‐induced iNKT cell activation and loss during viral infections.     

Antibody‐Mediated T‐Cell Reduction or Increased Levels of Chimerism Overcome Resistance to Cyclophosphamide‐Induced Tolerance in NKT‐Deficient Mice

We reported that invariant NKT‐cell knockout (iNKT KO) mice are resistant to the induction of intrathymic chimerism and clonal deletion in the cyclophosphamide (CP)‐induced tolerance system (CPS). However, another report shows that clonal deletion with chimerism may be intact in iNKT KO recipients in a bone marrow transplantation model. We also reported that pretreatment with anti‐Thy1.2 mAb, which reduces the number of T cells and iNKT cells, promotes allograft tolerance across H‐2 barriers in the CPS. In this study, we evaluated the efficacy of T‐cell depletion in the CPS, and the relationship between the role played by iNKT cells in central tolerance and mixed chimerism. BALB/c (H‐2^d) wild‐type, or iNKT KO (Jα18^?/?) mice were pretreated with 20–100 μg of anti‐Thy1.2 mAb and given 10⁸ donor DBA/2 (H‐2^d) spleen cells on Day 0, and 200 mg/kg CP on Day 2. Pretreatment with T‐cell depletion resulted in higher levels of mixed chimerism, increased intrathymic clonal deletion of donor‐reactive cells, and the induction of skin graft tolerance in iNKT KO recipients in CPS. This suggests that the high levels of mixed chimerism overcame the resistance to CP‐induced tolerance in iNKT KO mice. Consistently, the enhancement of mixed chimerism by injection of tolerant donor spleen cells (SC) rendered iNKT KO recipients susceptible to CP‐induced tolerance. These results suggest that iNKT‐cell‐mediated immunoregulation of central tolerance is evident at low levels of peripheral mixed chimerism in the CPS.     

B cell‐intrinsic MyD88 signaling controls IFN‐γ‐mediated early IgG2c class switching in mice in response to a particulate adjuvant

Adjuvants improve the potency of vaccines, but the modes of action (MOAs) of most adjuvants are largely unknown. TLR‐dependent and ‐independent innate immune signaling through the adaptor molecule MyD88 has been shown to be pivotal to the effects of most adjuvants; however, MyD88's involvement in the TLR‐independent MOAs of adjuvants is poorly understood. Here, using the T‐dependent antigen NIPOVA and a unique particulate adjuvant called synthetic hemozoin (sHZ), we show that MyD88 is required for early GC formation and enhanced antibody class‐switch recombination (CSR) in mice. Using cell‐type‐specific MyD88 KO mice, we found that IgG2c class switching, but not IgG1 class switching, was controlled by B cell‐intrinsic MyD88 signaling. Notably, IFN‐γ produced by various cells including T cells, NK cells, and dendritic cells was the primary cytokine for IgG2c CSR and B‐cell intrinsic MyD88 is required for IFN‐γ production. Moreover, IFN‐γ receptor (IFNγR) deficiency abolished sHZ‐induced IgG2c production, while recombinant IFN‐γ administration successfully rescued IgG2c CSR impairment in mice lacking B‐cell intrinsic MyD88. Together, our results show that B cell‐intrinsic MyD88 signaling is involved in the MOA of certain particulate adjuvants and this may enhance our specific understanding of how adjuvants and vaccines work.     

Intrinsic T‐ and B‐cell defects impair T‐cell‐dependent antibody responses in mice lacking the actin‐bundling protein L‐plastin

Germinal center development, critical for long‐term humoral immunity, requires the trafficking of T and B lymphocytes to defined tissues and locations after antigenic challenge. The molecular mechanisms that support lymphocyte trafficking through the linkage of extracellular chemotactic and adhesive cues to the actin cytoskeleton are not yet fully defined. We have previously identified the actin‐bundling protein L‐plastin (LPL) as a requisite intermediary in both naive B and T lymphocyte migration and in T‐cell activation. We tested the hypothesis that humoral immunity would require LPL. We show that mice lacking LPL demonstrated defective germinal center formation and reduced production of T‐cell‐dependent antibodies. T cells from LPL^?/? mice exhibited defective expansion of the follicular helper T population. Reduced expansion of LPL^?/? follicular helper T cells correlated with impaired trafficking to or retention of cells in the spleen following challenge, highlighting the importance of initial lymphocyte recruitment to the eventual success of the immune response. Furthermore, LPL^?/? B cells demonstrated cell‐intrinsic defects in population expansion and in differentiation into germinal center B cells. LPL thus modulates both T‐ and B‐cell function during the germinal center reaction and the production of T‐cell‐dependent antibody responses.     

Skewed B cell differentiation affects lymphoid organogenesis but not T cell‐mediated autoimmunity

B cell receptor (BCR) signalling determines B cell differentiation and may potentially alter T cell‐mediated immune responses. In this study we used two transgenic strains of BCR‐deficient mice expressing Epstein–Barr virus latent membrane protein (LMP)2A in B cells, where either follicular and marginal zone differentiation (D_HLMP2A mice) or B‐1 cell development (V_HLMP2A mice) were supported, and evaluated the effects of skewed B lymphocyte differentiation on lymphoid organogenesis and T cell responses in vivo. Compared to wild‐type animals, both transgenic strains displayed alterations in the composition of lymphoid organs and in the dynamics of distinct immune cell subsets following immunization with the self‐antigen PLP_185–206. However, ex‐vivo T cell proliferation to PLP_185–206 peptide measured in immunized D_HLMP2A and V_HLMP2A mice was similar to that detected in immunized control mice. Further, clinical expression of experimental autoimmune encephalitis in both LMP2A strains was identical to that of wild‐type mice. In conclusion, mice with skewed B cell differentiation driven by LMP2A expression in BCR‐negative B cells do not show changes in the development of a T cell mediated disease model of autoimmunity, suggesting that compensatory mechanisms support the generation of T cell responses.     

IFN‐γ‐STAT1 signal regulates the differentiation of inducible Treg: Potential role for ROS‐mediated apoptosis

Regulatory CD4⁺ T cells are important for the homeostasis of immune cells, and their absence correlates with autoimmune disorders. However, how the immune system regulates Treg homeostasis remains unclear. We found that IFN‐γ‐deficient‐mice had more forkhead box P3 (FOXP3⁺) cells than WT mice in all secondary lymphoid organs except the thymus. However, T‐bet‐ or IL‐4Rα‐deficient mice did not show a similar increase. In vitro differentiation studies showed that conversion of naïve T cells into FOXP3⁺ cells (neo‐generated inducible Treg (iTreg)) by TGF‐β was significantly inhibited by IFN‐γ in a STAT‐1‐dependent manner. Moreover, an in vivo adoptive transfer study showed that inhibition of FOXP3⁺ iTreg generation by IFN‐γ was a T‐cell autocrine effect. This inhibitory effect of IFN‐γ on iTreg generation was significantly abrogated after N‐acetyl‐L ‐cysteine treatment both in vitro and in vivo, indicating that IFN‐γ regulation of iTreg generation is dependent on ROS‐mediated apoptosis. Therefore, our results suggest that autocrine IFN‐γ can negatively regulate the neo‐generation of FOXP3⁺ iTreg through ROS‐mediated apoptosis in the periphery.     

Differential requirement for CARMA1 in agonist‐selected T‐cell development

Caspase recruitment domain‐containing membrane‐associated guanylate kinase protein‐1 (CARMA1) is a critical component of the NF‐κB signaling cascade mediated by TCR engagement. In addition to activation of naïve T cells, TCR signaling is important for the development of agonist‐selected T‐cell subsets such as Treg, NKT cells, and CD8‐αα T cells. However, little is known about the role of CARMA1 in the development of these lineages. Here we show that CARMA1‐deficient mice (CARMA1^?/?) have altered populations of specific subsets of agonist‐selected T cells. Specifically, CARMA1^?/? mice have impaired natural and adaptive Treg development, whereas NKT cell numbers are normal compared with wild‐type mice. Interestingly, CD8‐αα T cells, which may also be able to develop through an extrathymic selection pathway, are enriched in the gut of CARMA1^?/? mice, whereas memory‐phenotype CD4⁺ T cells (CD62L^low/CD44^high) are present at reduced numbers in the periphery. These results indicate that CARMA1 is essential for Treg development, but is not necessary for the development of other agonist‐selected T‐cell subsets. Overall, these data reveal an important but differential role for CARMA1‐mediated TCR signaling in T‐cell development.