Purification and analysis of kidney-infiltrating leukocytes in a mouse model of lupus nephritis.

Renal injury often occurs as a complication in autoimmune diseases such as systemic lupus erythematosus (SLE). It is estimated that a minimum of 20% SLE patients develop lupus nephritis, a condition that can be fatal when the pathology progresses to end-stage renal disease. Studies in animal models showed that incidence of immune cell infiltrates in the kidney was linked to pathological injury and correlated with severe lupus nephritis. Thus, preventing immune cell infiltration into the kidney is a potential approach to impede the progression to an end-stage disease. A requirement to investigate the role of kidney-infiltrating leukocytes is the development of reproducible and efficient protocols for purification and characterization of immune cells in kidney samples. This chapter describes a detailed methodology that discriminates tissue-resident leukocytes from blood-circulating cells that are found in kidney. Our protocol was designed to maximize cell viability and to reduce variability among samples, with a combination of intravascular staining and magnetic bead separation for leukocyte enrichment. Experiments included as example were performed with FcγRIIb[KO] mice, a well-characterized murine model of SLE. We identified T cells and macrophages as the primary leukocyte subsets infiltrating into the kidney during severe nephritis, and we extensively characterized them phenotypically by flow cytometry.

Autoantibodies inhibit Plasmodium falciparum growth and are associated with protection from clinical malaria.

Many infections, including malaria, are associated with an increase in autoantibodies (AAbs). Prior studies have reported an association between genetic markers of susceptibility to autoimmune disease and resistance to malaria, but the underlying mechanisms are unclear. Here, we performed a longitudinal study of children and adults (n = 602) in Mali and found that high levels of plasma AAbs before the malaria season independently predicted a reduced risk of clinical malaria in children during the ensuing malaria season. Baseline AAb seroprevalence increased with age and asymptomatic Plasmodium falciparum infection. We found that AAbs purified from the plasma of protected individuals inhibit the growth of blood-stage parasites and bind P. falciparum proteins that mediate parasite invasion. Protected individuals had higher plasma immunoglobulin G (IgG) reactivity against 33 of the 123 antigens assessed in an autoantigen microarray. This study provides evidence in support of the hypothesis that a propensity toward autoimmunity offers a survival advantage against malaria.

MAVS Positively Regulates Mitochondrial Integrity and Metabolic Fitness in B Cells.

Activated B cells experience metabolic changes that require mitochondrial remodeling, in a process incompletely defined. In this study, we report that mitochondrial antiviral signaling protein (MAVS) is involved in BCR-initiated cellular proliferation and prolonged survival. MAVS is well known as a mitochondrial-tethered signaling adaptor with a central role in viral RNA-sensing pathways that induce type I IFN. The role of MAVS downstream of BCR stimulation was recognized in absence of IFN, indicative of a path for MAVS activation that is independent of viral infection. Mitochondria of BCR-activated MAVS-deficient mouse B cells exhibited a damaged phenotype including disrupted mitochondrial morphology, excess mitophagy, and the temporal progressive blunting of mitochondrial oxidative capacity with mitochondrial hyperpolarization and cell death. Costimulation of MAVS-deficient B cells with anti-CD40, in addition to BCR stimulation, partially corrected the mitochondrial structural defects and functionality. Our data reveal a (to our knowledge) previously unrecognized role of MAVS in controlling the metabolic fitness of B cells, most noticeable in the absence of costimulatory help.

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

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.

Aedes aegypti sialokinin facilitates mosquito blood feeding and modulates host immunity and vascular biology.

Saliva from mosquitoes contains vasodilators that antagonize vasoconstrictors produced at the bite site. Sialokinin is a vasodilator present in the saliva of Aedes aegypti. Here, we investigate its function and describe its mechanism of action during blood feeding. Sialokinin induces nitric oxide release similar to substance P. Sialokinin-KO mosquitoes produce lower blood perfusion than parental mosquitoes at the bite site during probing and have significantly longer probing times, which result in lower blood feeding success. In contrast, there is no difference in feeding between KO and parental mosquitoes when using artificial membrane feeders or mice that are treated with a substance P receptor antagonist, confirming that sialokinin interferes with host hemostasis via NK1R signaling. While sialokinin-KO saliva does not affect virus infection in vitro, it stimulates macrophages and inhibits leukocyte recruitment in vivo. This work highlights the biological functionality of salivary proteins in blood feeding.

CCL17-producing cDC2s are essential in end-stage lupus nephritis and averted by a parasitic infection.

Lupus nephritis is a severe organ manifestation in systemic lupus erythematosus leading to kidney failure in a subset of patients. In lupus-prone mice, controlled infection with Plasmodium parasites protects against the progression of autoimmune pathology including lethal glomerulonephritis. Here, we demonstrate that parasite-induced protection was not due to a systemic effect of infection on autoimmunity as previously assumed, but rather to specific alterations in immune cell infiltrates into kidneys and renal draining lymph nodes. Infection of lupus-prone mice with a Plasmodium parasite did not reduce the levels or specificities of autoreactive antibodies, vasculitis, immune complex-induced innate activation, or hypoxia. Instead, infection uniquely reduced kidney-infiltrating CCL17-producing bone marrow-derived type 2 inflammatory dendritic cells (iDC2s). Bone marrow reconstitution experiments revealed that infection with Plasmodium caused alterations in bone marrow cells that hindered the ability of DC2s to infiltrate the kidneys. The essential role for CCL17 in lupus nephritis was confirmed by in vivo depletion with a blocking antibody, which reduced kidney pathology and immune infiltrates, while bypassing the need for parasitic infection. Therefore, infiltration into the kidneys of iDC2s, with the potential to prime local adaptive responses, is an essential regulated event in the transition from manageable glomerulonephritis to lethal tubular injury.

The E3 ubiquitin ligase MARCH1 regulates antimalaria immunity through interferon signaling and T cell activation.

Malaria infection induces complex and diverse immune responses. To elucidate the mechanisms underlying host-parasite interaction, we performed a genetic screen during early (24 h) Plasmodium yoelii infection in mice and identified a large number of interacting host and parasite genes/loci after transspecies expression quantitative trait locus (Ts-eQTL) analysis. We next investigated a host E3 ubiquitin ligase gene (March1) that was clustered with interferon (IFN)-stimulated genes (ISGs) based on the similarity of the genome-wide pattern of logarithm of the odds (LOD) scores (GPLS). March1 inhibits MAVS/STING/TRIF-induced type I IFN (IFN-I) signaling in vitro and in vivo. However, in malaria-infected hosts, deficiency of March1 reduces IFN-I production by activating inhibitors such as SOCS1, USP18, and TRIM24 and by altering immune cell populations. March1 deficiency increases CD86+DC (dendritic cell) populations and levels of IFN-γ and interleukin 10 (IL-10) at day 4 post infection, leading to improved host survival. T cell depletion reduces IFN-γ level and reverse the protective effects of March1 deficiency, which can also be achieved by antibody neutralization of IFN-γ. This study reveals functions of MARCH1 (membrane-associated ring-CH-type finger 1) in innate immune responses and provides potential avenues for activating antimalaria immunity and enhancing vaccine efficacy.

Transcriptional Control of Mature B Cell Fates.

The mature naïve B cell repertoire consists of three well-defined populations: B1, B2 (follicular B, FOB), and marginal zone B (MZB) cells. FOB cells are the dominant mature B cell population in the secondary lymphoid organs and blood of both humans and mice. The driving forces behind mature B lineage selection have been linked to B cell receptor (BCR) signaling strength and environmental cues, but how these fate-determination factors are transcriptionally regulated remains poorly understood. We summarize emerging data on the role of transcription factors (TFs) - particularly the ETS and IRF families - in regulating MZB and FOB lineage selection. Indeed, genomic analyses have identified four major groups of target genes that are crucial for FOB differentiation, revealing previously unrecognized pathways that ultimately determine biological responses specific to this lineage.

Antiviral Adaptor MAVS Promotes Murine Lupus With a B Cell Autonomous Role.

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by increased production of autoantibodies, which commonly target nuclear antigens, and concomitant deposition of immune complexes that cause inflammation in tissues. SLE is often associated with increased systemic expression of type I interferons, in some cases due to dysregulation in nucleic acid-sensing innate pathways. There is strong genetic evidence for a link between cytoplasmic RNA sensing pathways (RIG-I/MDA5) and SLE, both in human patients and murine models, however questions still remain regarding pathway initiation, cell types involved and downstream effects. Here we show that MAVS, an essential adaptor for RIG-I/MDA5 signaling, is necessary for all symptoms of autoimmune disease that develop spontaneously in the lupus model FcγRIIB-/- mice. This effect was independent of type I interferon signaling, TLR7 expression or STING, all three factors that have been connected to autoimmunity. Mixed bone marrow reconstitution experiments showed reduced occurrence in autoimmune germinal centers and diminished autoantibody production by MAVS-deficient B cells. Thus, MAVS plays a B cell intrinsic role in autoreactive B cell activation that is independent of its anti-viral functions and independent of elevated type I interferon expression.

Strong Selection of a Few Dominant CD8 Clones in a TLR7-Dependent Autoimmune Mouse Model.

Systemic lupus is characterized by the expansion of a self-reactive repertoire of B cells and CD4 cells that together promote IgG Ab production against common nuclear Ags. Although several studies have suggested roles for CD8+ T cells in lupus, the full contribution of these lymphocytes to disease remains undefined. In particular, few studies have examined TCR clonotypes of the CD8 pool in lupus. We previously described activated but nonpathogenic CD8+ T cells in a mouse model of systemic autoimmune disease triggered by increased copy number of the tlr7 gene (TLR7tg mice), in which some of these T cells accumulate in the brain. In this article, we report, through the analysis of TCRß sequences, that CD8 cells from TLR7tg animals are strongly selected for a small number of clones, some of them reaching 30% of the repertoire, compared with less than 0.4% for the top clone in any wild type mice. High frequency clones are variable in sequence among individual TLR7tg mice and are distinct from top clones in the control animals, whereas CDR3 sequences of spleen and brain-resident T cells from the same TLR7tg animals have perfect concordance. These results suggest that top CD8 clones are selected in stochastic fashion in each animal but limit further diversification, and that brain-infiltrating CD8 cells in TLR7tg mice are not selected by a common tissue Ag. This kind of extreme clonal dominance and narrowing of the CD8+ repertoire might impair anti-viral responses and should be considered as an additional detrimental feature of chronic autoimmune disease.

Transcription factors IRF8 and PU.1 are required for follicular B cell development and BCL6-driven germinal center responses.

The IRF and Ets families of transcription factors regulate the expression of a range of genes involved in immune cell development and function. However, the understanding of the molecular mechanisms of each family member has been limited due to their redundancy and broad effects on multiple lineages of cells. Here, we report that double deletion of floxed Irf8 and Spi1 (encoding PU.1) by Mb1-Cre (designated DKO mice) in the B cell lineage resulted in severe defects in the development of follicular and germinal center (GC) B cells. Class-switch recombination and antibody affinity maturation were also compromised in DKO mice. RNA-seq (sequencing) and ChIP-seq analyses revealed distinct IRF8 and PU.1 target genes in follicular and activated B cells. DKO B cells had diminished expression of target genes vital for maintaining follicular B cell identity and GC development. Moreover, our findings reveal that expression of B-cell lymphoma protein 6 (BCL6), which is critical for development of germinal center B cells, is dependent on IRF8 and PU.1 in vivo, providing a mechanism for the critical role for IRF8 and PU.1 in the development of GC B cells.

G-protein-coupled formyl peptide receptors play a dual role in neutrophil chemotaxis and bacterial phagocytosis.

A dogma of innate immunity is that neutrophils use G-protein-coupled receptors (GPCRs) for chemoattractant to chase bacteria through chemotaxis and then use phagocytic receptors coupled with tyrosine kinases to destroy opsonized bacteria via phagocytosis. Our current work showed that G-protein-coupled formyl peptide receptors (FPRs) directly mediate neutrophil phagocytosis. Mouse neutrophils lacking formyl peptide receptors (Fpr1/2-/-) are defective in the phagocytosis of Escherichia coli and the chemoattractant N-formyl-Met-Leu-Phe (fMLP)-coated beads. fMLP immobilized onto the surface of a bead interacts with FPRs, which trigger a Ca2+ response and induce actin polymerization to form a phagocytic cup for engulfment of the bead. This chemoattractant GPCR/Gi signaling works independently of phagocytic receptor/tyrosine kinase signaling to promote phagocytosis. Thus, in addition to phagocytic receptor-mediated phagocytosis, neutrophils also utilize the chemoattractant GPCR/Gi signaling to mediate phagocytosis to fight against invading bacteria.

T cell-dependent antigen adjuvanted with DOTAP-CpG-B but not DOTAP-CpG-A induces robust germinal center responses and high affinity antibodies in mice.

The development of vaccines for infectious diseases for which we currently have none, including HIV, will likely require the use of adjuvants that strongly promote germinal center responses and somatic hypermutation to produce broadly neutralizing antibodies. Here we compared the outcome of immunization with the T-cell dependent antigen, NP-conjugated to chicken gamma globulin (NP-CGG) adjuvanted with the toll-like receptor 9 (TLR9) ligands, CpG-A or CpG-B, alone or conjugated with the cationic lipid carrier, DOTAP. We provide evidence that only NP-CGG adjuvanted with DOTAP-CpG-B was an effective vaccine in mice resulting in robust germinal center responses, isotype switching and high affinity NP-specific antibodies. The effectiveness of DOTAP-CpG-B as an adjuvant was dependent on the expression of the TLR9 signaling adaptor MyD88 in immunized mice. These results indicate DOTAP-CpG-B but not DOTAP-CpG-A is an effective adjuvant for T cell-dependent protein antigen-based vaccines.

B Cells Produce Type 1 IFNs in Response to the TLR9 Agonist CpG-A Conjugated to Cationic Lipids.

B cells express the innate receptor, TLR9, which signals in response to unmethylated CpG sequences in microbial DNA. Of the two major classes of CpG-containing oligonucleotides, CpG-A appears restricted to inducing type 1 IFN in innate immune cells and CpG-B to activating B cells to proliferate and produce Abs and inflammatory cytokines. Although CpGs are candidates for adjuvants to boost innate and adaptive immunity, our understanding of the effect of CpG-A and CpG-B on B cell responses is incomplete. In this study we show that both CpG-B and CpG-A activated B cells in vitro to proliferate, secrete Abs and IL-6, and that neither CpG-B nor CpG-A alone induced type 1 IFN production. However, when incorporated into the cationic lipid, DOTAP, CpG-A, but not CpG-B, induced a type 1 IFN response in B cells in vitro and in vivo. We provide evidence that differences in the function of CpG-A and CpG-B may be related to their intracellular trafficking in B cells. These findings fill an important gap in our understanding of the B cell response to CpGs, with implications for the use of CpG-A and CpG-B as immunomodulators.

Expanding the B Cell-Centric View of Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by a breakdown of self-tolerance in B cells and the production of antibodies against nuclear self-antigens. Increasing evidence supports the notion that additional cellular contributors beyond B cells are important for lupus pathogenesis. In this review we consider recent advances regarding both the pathogenic and the regulatory role of lymphocytes in SLE beyond the production of IgG autoantibodies. We also discuss various inflammatory effector cell types involved in cytokine production, removal of self-antigens, and responses to autoreactive IgE antibodies. We aim to integrate these ideas to expand the current understanding of the cellular components that contribute to disease progression and ultimately help in the design of novel, targeted therapeutics.

Non-pathogenic tissue-resident CD8+ T cells uniquely accumulate in the brains of lupus-prone mice.

Severe lupus often includes psychiatric and neurological sequelae, although the cellular contributors to CNS disease remain poorly defined. Using intravascular staining to discriminate tissue-localized from blood-borne cells, we find substantial accumulation of CD8+ T cells relative to other lymphocytes in brain tissue, which correlates with lupus disease and limited neuropathology. This is in contrast to all other affected organs, where infiltrating CD4+ cells are predominant. Brain-infiltrating CD8+ T cells represent an activated subset of those found in the periphery, having a resident-memory phenotype (CD69+CD122-PD1+CD44+CD62L-) and expressing adhesion molecules (VLA-4+LFA-1+) complementary to activated brain endothelium. Remarkably, infiltrating CD8+ T cells do not cause tissue damage in lupus-prone mice, as genetic ablation of these cells via ß2 m deficiency does not reverse neuropathology, but exacerbates disease both in the brain and globally despite decreased serum IgG levels. Thus, lupus-associated inflammation disrupts the blood-brain barrier in a discriminating way biased in favor of non-pathogenic CD8+ T cells relative to other infiltrating leukocytes, perhaps preventing further tissue damage in such a sensitive organ.

The Csk-Associated Adaptor PAG Inhibits Effector T Cell Activation in Cooperation with Phosphatase PTPN22 and Dok Adaptors.

The transmembrane adaptor PAG (Cbp) has been proposed to mediate membrane recruitment of Csk, a cytoplasmic protein tyrosine kinase playing a critical inhibitory role during T cell activation, by inactivating membrane-associated Src kinases. However, this model has not been validated by genetic evidence. Here, we demonstrate that PAG-deficient mice display enhanced T cell activation responses in effector, but not in naive, T cells. PAG-deficient mice also have augmented T cell-dependent autoimmunity and greater resistance to T cell anergy. Interestingly, in the absence of PAG, Csk becomes more associated with alternative partners; i.e., phosphatase PTPN22 and Dok adaptors. Combining PAG deficiency with PTPN22 or Dok adaptor deficiency further enhances effector T cell responses. Unlike PAG, Cbl ubiquitin ligases inhibit the activation of naive, but not of effector, T cells. Thus, Csk-associating PAG is a critical component of the inhibitory machinery controlling effector T cell activation in cooperation with PTPN22 and Dok adaptors.

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

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 Plasmodium yoelii nigeriensis N67 and C57BL/6 mouse model, we showed that infected CD40-/- mice had reduced STING and serum IFN-ß levels day-2 post infection, higher day-4 parasitemia, and earlier deaths. CD40 could greatly enhance STING-stimulated luciferase signals driven by the IFN-ß promoter in vitro, 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-ß 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.

Genome-wide Analysis of Host-Plasmodium yoelii Interactions Reveals Regulators of the Type I Interferon Response.

Invading pathogens trigger specific host responses, an understanding of which might identify genes that function in pathogen recognition and elimination. In this study, we performed trans-species expression quantitative trait locus (ts-eQTL) analysis using genotypes of the Plasmodium yoelii malaria parasite and phenotypes of mouse gene expression. We significantly linked 1,054 host genes to parasite genetic loci (LOD score ≥ 3.0). Using LOD score patterns, which produced results that differed from direct expression-level clustering, we grouped host genes that function in related pathways, allowing functional prediction of unknown genes. As a proof of principle, 14 of 15 randomly selected genes predicted to function in type I interferon (IFN-I) responses were experimentally validated using overexpression, small hairpin RNA knockdown, viral infection, and/or infection of knockout mice. This study demonstrates an effective strategy for studying gene function, establishes a functional gene database, and identifies regulators in IFN-I pathways.