Commensal bacteria direct selective cargo sorting to promote symbiosis.

Mucosal immunity protects a host from intestinal inflammation and infection and is profoundly influenced by symbiotic bacteria. Here we report that in mice symbiotic bacteria directed selective cargo sorting in Paneth cells to promote symbiosis through Nod2, a cytosolic bacterial sensor, and the multifunctional protein kinase LRRK2, both encoded by inflammatory bowel disease (IBD)-associated genes. Commensals recruited Nod2 onto lysozyme-containing dense core vesicles (DCVs), which was required for DCV localization of LRRK2 and a small GTPase, Rab2a. Deficiency of Nod2, LRRK2 or Rab2a or depletion of commensals resulted in lysosomal degradation of lysozyme. Thus, commensal bacteria and host factors orchestrate the lysozyme-sorting process to protect the host from enteric infection, implicating Paneth cell dysfunction in IBD pathogenesis.

Toll-like Receptor 4 Engagement on Dendritic Cells Restrains Phago-Lysosome Fusion and Promotes Cross-Presentation of Antigens.

The initiation of cytotoxic immune responses by dendritic cells (DCs) requires the presentation of antigenic peptides derived from phagocytosed microbes and infected or dead cells to CD8(+) T cells, a process called cross-presentation. Antigen cross-presentation by non-activated DCs, however, is not sufficient for the effective induction of immune responses. Additionally, DCs need to be activated through innate receptors, like Toll-like receptors (TLRs). During DC maturation, cross-presentation efficiency is first upregulated and then turned off. Here we show that during this transient phase of enhanced cross-presentation, phago-lysosome fusion was blocked by the topological re-organization of lysosomes into perinuclear clusters. LPS-induced lysosomal clustering, inhibition of phago-lysosome fusion and enhanced cross-presentation, all required expression of the GTPase Rab34. We conclude that TLR4 engagement induces a Rab34-dependent re-organization of lysosomal distribution that delays antigen degradation to transiently enhance cross-presentation, thereby optimizing the priming of CD8(+) T cell responses against pathogens.

B Cell Endosomal RAB7 Promotes TRAF6 K63 Polyubiquitination and NF-κB Activation for Antibody Class-Switching.

Upon activation by CD40 or TLR signaling, B lymphocytes activate NF-κB to induce activation-induced cytidine deaminase and, therefore, Ig class switch DNA recombination, as central to the maturation of the Ab and autoantibody responses. In this study, we show that NF-κB activation is boosted by colocalization of engaged immune receptors, such as CD40, with RAB7 small GTPase on mature endosomes, in addition to signals emanating from the receptors localized on the plasma membrane, in mouse B cells. In mature endosomes, RAB7 directly interacts with TRAF6 E3 ubiquitin ligase, which catalyzes K63 polyubiquitination for NF-κB activation. RAB7 overexpression in Cd19+/creRosa26fl-STOP-fl-Rab7 mouse B cells upregulates K63 polyubiquitination activity of TRAF6, enhances NF-κB activation and activation-induced cytidine deaminase induction, and boosts IgG Ab and autoantibody levels. This, together with the extensive intracellular localization of CD40 and the strong correlation of RAB7 expression with NF-κB activation in mouse lupus B cells, shows that RAB7 is an integral component of the B cell NF-κB activation machinery, likely through interaction with TRAF6 for the assembly of "intracellular membrane signalosomes."

Rab6b localizes to the Golgi complex in murine macrophages and promotes tumor necrosis factor release in response to mycobacterial infection.

The proinflammatory cytokine tumor necrosis factor (TNF) plays a central role in the host control of mycobacterial infections. Expression and release of TNF are tightly regulated, yet the molecular mechanisms that control the release of TNF by mycobacteria-infected host cells, in particular macrophages, are incompletely understood. Rab GTPases direct the transport of intracellular membrane-enclosed vesicles and are important regulators of macrophage cytokine secretion. Rab6b is known to be predominantly expressed in the brain where it functions in retrograde transport and anterograde vesicle transport for exocytosis. Whether it executes similar functions in the context of immune responses is unknown. Here we show that Rab6b is expressed by primary mouse macrophages, where it localized to the Golgi complex. Infection with Mycobacterium bovis bacille Calmette-Guérin (BCG) resulted in dynamic changes in Rab6b expression in primary mouse macrophages in vitro as well as in organs from infected mice in vivo. We further show that Rab6b facilitated TNF release by M. bovis BCG-infected macrophages, in the absence of discernible impact on Tnf messenger RNA and intracellular TNF protein expression. Our observations identify Rab6b as a positive regulator of M. bovis BCG-induced TNF trafficking and secretion by macrophages and positions Rab6b among the molecular machinery that orchestrates inflammatory cytokine responses by macrophages.

Rab32 GTPase, as a direct target of miR-30b/c, controls the intracellular survival of Burkholderia pseudomallei by regulating phagosome maturation.

Burkholderia pseudomallei is a gram-negative, facultative intracellular bacterium, which causes a disease known as melioidosis. Professional phagocytes represent a crucial first line of innate defense against invading pathogens. Uptake of pathogens by these cells involves the formation of a phagosome that matures by fusing with early and late endocytic vesicles, resulting in killing of ingested microbes. Host Rab GTPases are central regulators of vesicular trafficking following pathogen phagocytosis. However, it is unclear how Rab GTPases interact with B. pseudomallei to regulate the transport and maturation of bacterial-containing phagosomes. Here, we showed that the host Rab32 plays an important role in mediating antimicrobial activity by promoting phagosome maturation at an early phase of infection with B. pseudomallei. And we demonstrated that the expression level of Rab32 is increased through the downregulation of the synthesis of miR-30b/30c in B. pseudomallei infected macrophages. Subsequently, we showed that B. pseudomallei resides temporarily in Rab32-positive compartments with late endocytic features. And Rab32 enhances phagosome acidification and promotes the fusion of B. pseudomallei-containing phagosomes with lysosomes to activate cathepsin D, resulting in restricted intracellular growth of B. pseudomallei. Additionally, Rab32 mediates phagosome maturation depending on its guanosine triphosphate/guanosine diphosphate (GTP/GDP) binding state. Finally, we report the previously unrecognized role of miR-30b/30c in regulating B. pseudomallei-containing phagosome maturation by targeting Rab32 in macrophages. Altogether, we provide a novel insight into the host immune-regulated cellular pathway against B. pseudomallei infection is partially dependent on Rab32 trafficking pathway, which regulates phagosome maturation and enhances the killing of this bacterium in macrophages.

TBK-1 promotes autophagy-mediated antimicrobial defense by controlling autophagosome maturation.

Autophagy is a fundamental biological process of the eukaryotic cell contributing to diverse cellular and physiological functions including cell-autonomous defense against intracellular pathogens. Here, we screened the Rab family of membrane trafficking regulators for effects on autophagic elimination of Mycobacterium tuberculosis var. bovis BCG and found that Rab8b and its downstream interacting partner, innate immunity regulator TBK-1, are required for autophagic elimination of mycobacteria in macrophages. TBK-1 was necessary for autophagic maturation. TBK-1 coordinated assembly and function of the autophagic machinery and phosphorylated the autophagic adaptor p62 (sequestosome 1) on Ser-403, a residue essential for its role in autophagic clearance. A key proinflammatory cytokine, IL-1ß, induced autophagy leading to autophagic killing of mycobacteria in macrophages, and this IL-1ß activity was dependent on TBK-1. Thus, TBK-1 is a key regulator of immunological autophagy and is responsible for the maturation of autophagosomes into lytic bactericidal organelles.

TLR4 Receptor Induces 2-AG-Dependent Tolerance to Lipopolysaccharide and Trafficking of CB2 Receptor in Mast Cells.

Mast cells (MCs) contribute to the control of local inflammatory reactions and become hyporesponsive after prolonged TLR4 activation by bacterial LPS. The molecular mechanisms involved in endotoxin tolerance (ET) induction in MCs are not fully understood. In this study, we demonstrate that the endocannabinoid 2-arachidonoylglycerol (2-AG) and its receptor, cannabinoid receptor 2 (CB2), play a role in the establishment of ET in bone marrow-derived MCs from C57BL/6J mice. We found that CB2 antagonism prevented the development of ET and that bone marrow-derived MCs produce 2-AG in a TLR4-dependent fashion. Exogenous 2-AG induced ET similarly to LPS, blocking the phosphorylation of IKK and the p65 subunit of NF-κB and inducing the synthesis of molecular markers of ET. LPS caused CB2 receptor trafficking in Rab11-, Rab7-, and Lamp2-positive vesicles, indicating recycling and degradation of the receptor. 2-AG also prevented LPS-induced TNF secretion in vivo, in a MC-dependent model of endotoxemia, demonstrating that TLR4 engagement leads to 2-AG secretion, which contributes to the negative control of MCs activation. Our study uncovers a functional role for the endocannabinoid system in the inhibition of MC-dependent innate immune responses in vivo.

Rab9 defense against white spot syndrome virus by participation in autophagy in Marsupenaeus japonicas.

White spot syndrome virus (WSSV) is the main pathogen of shrimp and has led to considerable economic losses to the shrimp industry around the world. However, so far there are still no effective strategies to address this problem. In this paper, the tissue distribution of Rab9 as well as its defense mechanism against WSSV in Japanese shrimp (Marsupenaeus japonicas) was investigated. The results revealed that Rab9 had a higher expression in hemocyte and gill while expression was lower in heart, muscle, intestine, liver, indicating Rab9 was involved in the innate immune process. The results showed that the Rab9 expression increased when shrimp was challenged with WSSV compared with that of control, while the silence of Rab9 led to the increase of WSSV copies. In order to explore the antiviral mechanism of Rab9, it was demonstrated that the expression level of Rab9 changed during autophagy process, which indicated that Rab9 is participated in the autophagy procedure of shrimp. The fact that autophagy decreased after Rab9 silenced, may also suggest that Rab9 protein could affect autophagy. In short, the results showed Rab9 played a key role in antivirus through regulating autophagy. The results not only enlarge the limited views about molecular mechanism of Rab in invertebrate, but also help to enrich the immunological content in marine invertebrate.

Intracellular distribution and transcriptional regulation of Atlantic salmon (Salmo salar) Rab5c, 7a and 27a homologs by immune stimuli.

Rab GTPases control trafficking of intracellular vesicles and are key regulators of endocytic and secretory pathways. Due to their specific distribution, they may serve as markers for different endolysosomal compartments. Since Rab GTPases are involved in uptake and trafficking of endocytosed ligands and cell receptors, as well as secretion of immune mediators, they have been implicated in diverse immunological processes and their functions are often exploited by intracellular pathogens such as viruses. While Rab proteins have been studied extensively in mammals, their functions in vesicle trafficking in teleosts are not well known. In the present work, Atlantic salmon Rab5c, Rab7a and Rab27a homologs were studied in terms of intracellular distribution and gene expression. Structured illumination microscopy demonstrated that transgenic, GFP-tagged salmon Rab5c and Rab7a are, predominantly, located within early endosomes and late endosomes/lysosomes, respectively. In contrast, Rab27a showed a broader distribution, which indicates that it associates with diverse intracellular vesicles and organelles. Infection of salmon with Salmonid alphavirus subtype 3 (SAV3) enhanced the mRNA levels of all of the studied Rab isoforms in heart and head kidney and most of them were upregulated in spleen. This may reflect the capacity of the virus to exploit the functions of these rab proteins. It is also possible that the transcriptional regulation of Rab proteins in SAV3-infected organs may play a role in the antiviral immune response. The latter was further supported by in vitro experiments with adherent head kidney leukocytes. The expression of Rab5c and Rab27a was upregulated in these cells following stimulation with TLR ligands including CpG oligonucleotides and polyI:C. The expression of most of the analyzed Rab isoforms in the primary leukocytes was also enhanced by stimulation with type I IFN. Interestingly, IFN-gamma had a negative effect on Rab7a expression which may be linked to the priming activity of this cytokine on monocytes and macrophages. Overall, these data demonstrate that the intracellular distribution of Rab5c, Rab7a and Rab27a is phylogenetically conserved within vertebrates and that these molecules might be implicated in viral infections and the regulation of the antiviral immune response in Atlantic salmon.

Immune evasion by Salmonella: exploiting the VPAC1/VIP axis in human monocytes.

Immune evasion is a critical survival mechanism for bacterial colonization of deeper tissues and may lead to life-threatening conditions such as endotoxaemia and sepsis. Understanding these immune evasion pathways would be an important step for the development of novel anti-microbial therapeutics. Here, we report a hitherto unknown mechanism by which Salmonella exploits an anti-inflammatory pathway in human immune cells to obtain survival advantage. We show that Salmonella enterica serovar Typhimurium strain 4/74 significantly (P < 0·05) increased expression of mRNA and surface protein of the type 1 receptor (VPAC1) for anti-inflammatory vasoactive intestinal peptide (VIP) in human monocytes. However, we also show that S. Typhimurium induced retrograde recycling of VPAC1 from early endosomes to Rab11a-containing sorting endosomes, associated with the Golgi apparatus, and anterograde trafficking via Rab3a and calmodulin 1. Expression of Rab3a and calmodulin 1 were significantly increased by S. Typhimurium infection and W-7 (calmodulin antagonist) decreased VPAC1 expression on the cell membrane while CALP-1 (calmodulin agonist) increased VPAC1 expression (P < 0·05). When infected monocytes were co-cultured with VIP, a significantly higher number of S. Typhimurium were recovered from these monocytes, compared with S. Typhimurium recovered from monocytes cultured only in cell media. We conclude that S. Typhimurium infection exploits host VPAC1/VIP to gain survival advantage in human monocytes.

Rab7 controls innate immunity by regulating phagocytosis and antimicrobial peptide expression in Chinese mitten crab.

The Rab family is the most significant subfamily of small GTP-binding proteins. These proteins have widespread intracellular localization and play an important role in many biological processes. Rab7 plays a crucial role in the innate immune system of crustaceans. In the present study, we cloned and characterized Rab7 from Chinese mitten crab (Eriocheir sinensis), designated EsRab7. The full-length of the EsRab7 cDNA sequence is 1,257 bp and contains a 618-bp open reading frame encoding a 205-amino acid polypeptide. Bioinformatics analysis showed that the Rab7 protein was highly conserved during evolution. Quantitative real-time PCR showed the highest tissue expression in muscle, followed by hepatopancreas. EsRab7 was significantly upregulated in hemocytes after stimulation by Gram-positive Staphylococcus aureus or Gram-negative Vibrio parahaemolyticus. Further studies showed that EsRab7 knockdown during bacterial stimulation resulted in decreased bacterial phagocytosis. In addition, EsRab7 regulated the expression of antimicrobial peptides via the Toll signaling pathway. Collectively, these results demonstrate that EsRab7 plays critical roles in antimicrobial function in the Chinese mitten crab.

Rab11-FIP3 Regulation of Lck Endosomal Traffic Controls TCR Signal Transduction.

The role of endosomes in receptor signal transduction is a long-standing question, which remains largely unanswered. The T cell Ag receptor and various components of its proximal signaling machinery are associated with distinct endosomal compartments, but how endosomal traffic affects T cell signaling remains ill-defined. In this article, we demonstrate in human T cells that the subcellular localization and function of the protein tyrosine kinase Lck depends on the Rab11 effector FIP3 (Rab11 family interacting protein-3). FIP3 overexpression or silencing and its ability to interact with Rab11 modify Lck subcellular localization and its delivery to the immunological synapse. Importantly, FIP3-dependent Lck localization controls early TCR signaling events, such as tyrosine phosphorylation of TCRζ, ZAP70, and LAT and intracellular calcium concentration, as well as IL-2 gene expression. Interestingly, FIP3 controls both steady-state and poststimulation phosphotyrosine and calcium levels. Finally, our findings indicate that FIP3 modulates TCR-CD3 cell surface expression via the regulation of steady-state Lck-mediated TCRζ phosphorylation, which in turn controls TCRζ protein levels. This may influence long-term T cell activation in response to TCR-CD3 stimulation. Therefore, our data underscore the importance of finely regulated endosomal traffic in TCR signal transduction and T cell activation leading to IL-2 production.

Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils.

There is compelling evidence for the role of the leucine-rich repeat kinase 2 (LRRK2) and in particular its kinase function in Parkinson's disease. Orally bioavailable, brain penetrant and potent LRRK2 kinase inhibitors are in the later stages of clinical development. Here, we describe a facile and robust assay to quantify LRRK2 kinase pathway activity by measuring LRRK2-mediated phosphorylation of Rab10 in human peripheral blood neutrophils. We use the selective MJFF-pRab10 monoclonal antibody recognising the Rab10 Thr73 phospho-epitope that is phosphorylated by LRRK2. We highlight the feasibility and practicability of using our assay in the clinical setting by studying a few patients with G2019S LRRK2 associated and sporadic Parkinson's as well as healthy controls. We suggest that peripheral blood neutrophils are a valuable resource for LRRK2 research and should be considered for inclusion in Parkinson's bio-repository collections as they are abundant, homogenous and express relatively high levels of LRRK2 as well as Rab10. In contrast, the widely used peripheral blood mononuclear cells are heterogeneous and only a minority of cells (monocytes and contaminating neutrophils) express LRRK2. While our LRRK2 kinase pathway assay could assist in patient stratification based on LRRK2 kinase activity, we envision that it may find greater utility in pharmacodynamic and target engagement studies in future LRRK2 inhibitor trials.

Endothelial Cell Inflammation and Barriers Are Regulated by the Rab26-Mediated Balance between ß2-AR and TLR4 in Pulmonary Microvessel Endothelial Cells.

Rab26 GTPase modulates the trafficking of cell surface receptors, such as G protein-coupled receptors including α2-adrenergic receptors in some cell types. However, the effect of Rab26 on ß2-adrenergic receptor (ß2-AR) trafficking or/and Toll-like receptor 4 (TLR4) expression in human pulmonary microvascular endothelial cells (HPMECs) is still unclear. Here, we investigated the role of Rab26 in regulating the expression of ß2-ARs and TLR4 in HPMECs and the effect of these receptors' imbalance on endothelial cell barrier function. The results showed that there was unbalance expression in these receptors, where ß2-AR expression was remarkably reduced, and TLR4 was increased on the cell membrane after lipopolysaccharide (LPS) treatment. Furthermore, we found that Rab26 overexpression not only upregulated ß2-ARs but also downregulated TLR4 expression on the cell membrane. Subsequently, the TLR4-related inflammatory response was greatly attenuated, and the hyperpermeability of HPMECs also was partially relived. Taken together, these data suggest that basal Rab26 maintains the balance between ß2-ARs and TLR4 on the cell surface, and it might be a potential therapeutic target for diseases involving endothelial barrier dysfunction.

Rab35 GTPase: A Central Regulator of Phosphoinositides and F-actin in Endocytic Recycling and Beyond.

Rab35 is one of the first discovered members of the large Rab GTPase family, yet it received little attention for 10 years being considered merely as a Rab1-like GTPase. In 2006, Rab35 was recognized as a unique Rab GTPase localized both at the plasma membrane and on endosomes, playing essential roles in endocytic recycling and cytokinesis. Since then, Rab35 has become one of the most studied Rabs involved in a growing number of cellular functions, including endosomal trafficking, exosome release, phagocytosis, cell migration, immunological synapse formation and neurite outgrowth. Recently, Rab35 has been acknowledged as an oncogenic GTPase with activating mutations being found in cancer patients. In this review, we provide a comprehensive summary of known Rab35-dependent cellular functions and detail the few Rab35 effectors characterized so far. We also review how the Rab35 GTP/GDP cycle is regulated, and emphasize a newly discovered mechanism that controls its tight activation on newborn endosomes. We propose that the involvement of Rab35 in such diverse and apparently unrelated cellular functions can be explained by the central role of this GTPase in regulating phosphoinositides and F-actin, both on endosomes and at the plasma membrane.

Small GTPase Rab8a-recruited Phosphatidylinositol 3-Kinase γ Regulates Signaling and Cytokine Outputs from Endosomal Toll-like Receptors.

LPS-mediated activation of Toll-like receptor 4 (TLR4) in macrophages results in the coordinated release of proinflammatory cytokines, followed by regulatory mediators, to ensure that this potentially destructive pathway is tightly regulated. We showed previously that Rab8a recruits PI3Kγ for Akt-dependent signaling during TLR4 activation to limit the production of the proinflammatory cytokines IL-6 and IL-12p40 while enhancing the release of the regulatory/anti-inflammatory cytokine IL-10. Here we broaden the array of immune receptors controlled by Rab8a-PI3Kγ and further define the Rab-mediated membrane domains required for signaling. With CRISPR/Cas9-mediated gene editing to stably knock out and recover Rab8a in macrophage cell lines, we match Akt signaling profiles with cytokine outputs, confirming that Rab8a is a novel regulator of the Akt/mammalian target of rapamycin (mTOR) pathway downstream of multiple TLRs. Upon developing a Rab8a activation assay, we show that TLR3 and 9 agonists also activate Rab8a. Live-cell imaging reveals that Rab8a is first recruited to the plasma membrane and dorsal ruffles, but it is retained during collapse of ruffles to form macropinosomes enriched for phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), suggesting that the macropinosome is the location where Rab8a is active. We pinpoint macropinosomes as the sites for Rab8-mediated biasing of inflammatory signaling responses via inducible production of anti-inflammatory cytokines. Thus, Rab8a and PI3Kγ are positioned in multiple TLR pathways, and this signaling axis may serve as a pharmacologically tractable target during infection and inflammation.

TLR sorting by Rab11 endosomes maintains intestinal epithelial-microbial homeostasis.

Compartmentalization of Toll-like receptors (TLRs) in intestinal epithelial cells (IECs) regulates distinct immune responses to microbes; however, the specific cellular machinery that controls this mechanism has not been fully identified. Here we provide genetic evidences that the recycling endosomal compartment in enterocytes maintains a homeostatic TLR9 intracellular distribution, supporting mucosal tolerance to normal microbiota. Genetic ablation of a recycling endosome resident small GTPase, Rab11a, a gene adjacent to a Crohn's disease risk locus, in mouse IECs and in Drosophila midgut caused epithelial cell-intrinsic cytokine production, inflammatory bowel phenotype, and early mortality. Unlike wild-type controls, germ-free Rab11a-deficient mouse intestines failed to tolerate the intraluminal stimulation of microbial agonists. Thus, Rab11a endosome controls intestinal host-microbial homeostasis at least partially via sorting TLRs.

A Heterozygous RAB27A Mutation Associated with Delayed Cytolytic Granule Polarization and Hemophagocytic Lymphohistiocytosis.

Frequently fatal, primary hemophagocytic lymphohistiocytosis (HLH) occurs in infancy resulting from homozygous mutations in NK and CD8 T cell cytolytic pathway genes. Secondary HLH presents after infancy and may be associated with heterozygous mutations in HLH genes. We report two unrelated teenagers with HLH and an identical heterozygous RAB27A mutation (c.259G→C). We explore the contribution of this Rab27A missense (p.A87P) mutation on NK cell cytolytic function by cloning it into a lentiviral expression vector prior to introduction into the human NK-92 cell line. NK cell degranulation (CD107a expression), target cell conjugation, and K562 target cell lysis was compared between mutant- and wild-type-transduced NK-92 cells. Polarization of granzyme B to the immunologic synapse and interaction of mutant Rab27A (p.A87P) with Munc13-4 were explored by confocal microscopy and proximity ligation assay, respectively. Overexpression of the RAB27A mutation had no effect on cell conjugate formation between the NK and target cells but decreased NK cell cytolytic activity and degranulation. Moreover, the mutant Rab27A protein decreased binding to Munc13-4 and delayed granzyme B polarization toward the immunologic synapse. This heterozygous RAB27A mutation blurs the genetic distinction between primary and secondary HLH by contributing to HLH via a partial dominant-negative effect.

Rab12 Regulates Retrograde Transport of Mast Cell Secretory Granules by Interacting with the RILP-Dynein Complex.

Secretory granule (SG) transport is a critical step in regulated exocytosis including degranulation of activated mast cells. The latter process results in the release of multiple inflammatory mediators that play key roles in innate immunity, as well as in allergic responses. In this study, we identified the small GTPase Rab12 as a novel regulator of mast cell SG transport, and we provide mechanistic insights into its mode of action. We show that Rab12 is activated in a stimulus-dependent fashion and promotes microtubule-dependent retrograde transport of the SGs in the activated cells. We also show that this minus end transport of the SGs is mediated by the RILP-dynein complex and identify RILP as a novel effector of Rab12. Finally, we show that Rab12 negatively regulates mast cell degranulation. Taken together, our results identify Rab12 as a novel regulator of mast cell responses and disclose for the first time, to our knowledge, the mechanism of retrograde transport of the mast cell SGs.

Small Molecule Inhibition of Rab7 Impairs B Cell Class Switching and Plasma Cell Survival To Dampen the Autoantibody Response in Murine Lupus.

IgG autoantibodies mediate pathology in systemic lupus patients and lupus-prone mice. In this study, we showed that the class-switched IgG autoantibody response in MRL/Faslpr/lpr and C57/Sle1Sle2Sle2 mice was blocked by the CID 1067700 compound, which specifically targeted Ras-related in brain 7 (Rab7), an endosome-localized small GTPase that was upregulated in activated human and mouse lupus B cells, leading to prevention of disease development and extension of lifespan. These were associated with decreased IgG-expressing B cells and plasma cells, but unchanged numbers and functions of myeloid cells and T cells. The Rab7 inhibitor suppressed T cell-dependent and T cell-independent Ab responses, but it did not affect T cell-mediated clearance of Chlamydia infection, consistent with a B cell-specific role of Rab7. Indeed, B cells and plasma cells were inherently sensitive to Rab7 gene knockout or Rab7 activity inhibition in class switching and survival, respectively, whereas proliferation/survival of B cells and generation of plasma cells were not affected. Impairment of NF-κB activation upon Rab7 inhibition, together with the rescue of B cell class switching and plasma cell survival by enforced NF-κB activation, indicated that Rab7 mediates these processes by promoting NF-κB activation, likely through signal transduction on intracellular membrane structures. Thus, a single Rab7-inhibiting small molecule can target two stages of B cell differentiation to dampen the pathogenic autoantibody response in lupus.