Eukaryotic cells have membrane bound organelles that are essential for maintaining cellular organization and performing highly dynamic and specialized functions. These processes, which depend on the transfer and exchange of cargo between different organelles, require communication within cells and between cells and their environment, while maintaining the distinct identities of these compartments. Regulated transport and trafficking of intracellular vesicles is required to achieve these highly coordinated and spatiotemporally regulated events (for a comprehensive review see reference Stenmark, 2009). In this context, intracellular trafficking and the immune function of cells are linked in multiple ways and this coordination is critical for dynamic and specialized immune defenses (Pei et al., 2012).

Firstly, intracellular trafficking regulates dynamic signaling-dependent immune responses. During microbial infections, pathogen recognition by specific receptors leads to signaling events that trigger appropriate immune responses. Interestingly, the activation of receptors by microbial ligands can result in completely different responses depending on the localisation of these receptors. The best characterized group of receptors that control dynamic signaling is the Toll-like receptors (TLR). Activation of these pattern recognition receptors on the plasma membrane leads to different signals than when the receptors are activated by microbial components localized in the lumen of endocytic vesicles (Gay et al., 2014).

Secondly, innate immunity is driven in specific cell types by different intracellular pathways, including the uptake of macromolecules, apoptotic cells, pathogens and pathogen derived vesicles. The general “inbound” trafficking of macromolecules into plasma membrane-derived vesicles occurs via the endocytic pathway (Conner and Schmid, 2003). Depending on the mechanism of uptake and the cargo being internalized, endocytosis is broadly categorized as phagocytosis, which is restricted to certain cell types, or as pinocytosis, which is performed by all cell types (Conner and Schmid, 2003; Huotari and Helenius, 2011).

Thirdly, lysosome-mediated microbial degradation is required for the activation of the antigen-specific adaptive immune responses, which provides long-lasting immunity (for review, see reference Iwasaki and Medzhitov, 2015). The processing of antigens by immune cells relies on the endocytic and phagocytic pathways, where antigens are degraded and loaded on receptors that are then exposed on the cellular surface. Once internalized, endolysosomal trafficking contributes to the degradation of the cargo for antigen presentation, which then primes the adaptive immune responses (Vyas et al., 2008).

Finally, along with mechanisms for internalizing cargo, cells have constitutive and regulated exocytic pathways responsible for secreting molecules, including cytokines, hormones and neurotransmitters (Gundelfinger et al., 2003). As the regulated secretion of cytokines and immune mediators is critical during immunity and inflammation, this cellular pathway plays a critical role in these processes.

The Rab (Ras related in brain) family of small GTPases regulates vesicular transport and determines organelle identities, thereby functioning as central players in regulating the intracellular and cell-cell communication required to generate and maintain cellular homeostasis (Zerial and McBride, 2001; Stenmark, 2009). Rab GTPases act as molecular switches that localize to distinct intracellular membranes and regulate intracellular trafficking at the level of vesicle budding, motility, tethering, docking and fusion through their interactions with specific effectors (Zerial and McBride, 2001; Stenmark, 2009). Therefore, Rab proteins act as scaffolds that integrate signaling and trafficking events, providing spatio-temporal control of organelle maintenance and trafficking (Schwartz et al., 2007). Given their critical role in regulating intracellular trafficking, Rab GTPases modulate immune responses by regulating the transport of immune receptors (Husebye et al., 2010), the secretion of chemokines and cytokines (Murray et al., 2005) and by up-regulating the critical immune surveillance processes of endocytosis and phagocytosis (Stenmark, 2009; Flannagan et al., 2012; Figure 1).

Conserved microbe associated signatures, collectively referred to as pathogen-associated molecular patterns (PAMPs) are recognized by pattern recognition receptors (PRRs) on the surface of immune cells. This recognition induces intracellular signaling pathways responsible for inflammatory immune responses (Iwasaki and Medzhitov, 2015). In order to mount appropriate responses while avoiding chronic inflammation, intracellular trafficking must be tightly regulated in immune cells. Indeed, regulation occurs at least at two levels, including trafficking of PRRs and secretion of immune modulators (Schwartz et al., 2007). Phagocytes, namely macrophages, dendritic cells (DCs) and neutrophils, are critical components that drive the innate immune response. These cells engulf and destroy invading pathogens and drive customized adaptive immune responses (Iwasaki and Medzhitov, 2015). This process of pathogen uptake and destruction requires the concerted efforts of several members of the Rab family of small GTPases (Stenmark, 2009).

Innate immune responses induced by pathogen recognition instruct subsequent long lasting adaptive immunity mediated by antigen responsive B and T lymphocytes (Iwasaki and Medzhitov, 2015). To initiate cytotoxic immune responses, T cells must be “activated” by the process of antigen presentation. In antigen presenting cells (APC), pathogens or dead cells internalized by phagocytosis and macropinocytosis are first degraded in phagosomes and endosomes, and subsequently degraded antigens coupled to specific membrane receptors are transported by vesicles to the cell surface (Iwasaki and Medzhitov, 2015). It is therefore not surprising that Rab GTPases play a crucial role during antigen presentation and T cell mediated immunity in APC such as DCs and macrophages (Trombetta and Mellman, 2005). In order to mount an effective T cell response, it is important that antigen processing does not lead to antigen degradation. Therefore, as a strategy to reduce antigen degradation and to drive adaptive responses, Rab GTPase-dependent trafficking contributes to slower acidification and phagosome maturation in DCs (Savina and Amigorena, 2007). Rab27a-dependent trafficking of LROs causes the recruitment of the NADPH oxidase subunit NOX2 to phagosomes (Jancic et al., 2007), where it contributes to slower antigen processing by increasing phagosomal pH (Savina et al., 2006) and reducing phagosomal proteolysis by affecting cathepsins (Rybicka et al., 2012). Rab34 interacts with RILP to regulate lysosomal positioning and fusion with phagosomes (Cantalupo et al., 2001; Wang and Hong, 2002; Kasmapour et al., 2012). Toll-like receptor 4 (TLR4) engagement on DCs in response to LPS stimulation causes Rab34-dependent lysosomal clustering thereby delaying phagosomal maturation and antigen degradation and allowing for better T cell priming (Alloatti et al., 2015).

Phagosomes containing microbial components that engage TLR signaling recruit major histocompatibility complex (MHC) class I molecules from Rab11 positive endosomal recycling compartments (ERC) (Adiko et al., 2015). Rab11 contributes to antigen cross-presentation by trafficking and maintaining MHC class I molecules at the ERC (Nair-Gupta et al., 2014). Additionally, Rab11a has been shown to recruit TLR4 from ERC to bacteria-containing phagosomes, contributing to interferon regulatory factor-3 signaling and IFN-β production, further supporting its role in immune signaling (Husebye et al., 2010). Rab8a, Rab10, Rab7b are among the other Rab GTPases that can modulate TLR4-dependent immune responses (Wang et al., 2007, 2010; Luo et al., 2014).

While not directly implicated in antigen presentation by APCs, Rab9 was recently shown to mediate mitochondrial antigen presentation (Matheoud et al., 2016). In addition to their role in signaling and metabolic functions, mitochondria are important in immune responses and several PAMP-dependent signaling pathways require mitochondria-derived reactive oxygen species (for a comprehensive review, see reference Weinberg et al., 2015). Even though mitochondria are implicated in autoimmunity, the mechanisms responsible for recognition of self-antigens to develop immune tolerance are not well known (Weinberg et al., 2015). Damaged mitochondria are eliminated by mitophagy that in turn limits the presentation of mitochondrial antigens. Recent work has shown the presence of mitochondria derived vesicles (MDVs), which serve as a cellular quality control mechanism whereby damaged mitochondrial components like lipids and outer membrane can be delivered to peroxisomes for degradation (Neuspiel et al., 2008). These MDVs are important for mitochondrial antigen presentation and require Rab9 for their formation, while their fusion with lysosomes occurs in a Rab7-dependent manner (Matheoud et al., 2016).

Several studies on immune cells have provided evidence for the transcriptional control of intracellular membrane trafficking proteins. Collectively, these studies have demonstrated the role of immune modulators and microbes in regulating Rab protein expression (Pei et al., 2012). IFN-γ produced by natural killer and natural killer T cells is critical for immunity against viral and bacterial infections and contributes to macrophage activation by increasing phagocytosis and production of pro-inflammatory cytokines (Schoenborn and Wilson, 2007). Macrophages stimulated with IFN-γ show an increase in the expression of Rab5a and Rab20, both of which are important in phagosome maturation (Alvarez-Dominguez and Stahl, 1998; Vieira et al., 2003; Pei et al., 2014, 2015). Furthermore, the expression levels of Rab20 and Rab34 are up-regulated in response to M. avium and M. smegmatis infection, with Rab10 also up-regulated by M. smegmatis infection (Gutierrez et al., 2008).

Given the critical role of Rab5 in controlling both phagosome-early endosome fusion and the maturation of phagosomes into degradative compartments, cytokine-dependent regulation of Rab5 levels could provide control over phagosomal maturation. Along with upregulation through IFN-γ stimulation (Alvarez-Dominguez and Stahl, 1998), Rab5 expression can be up-regulated by interleukins (IL) 4 and 6. IL-4 induces alternate activation of macrophages and together with prostaglandin E2 (PGE2) induces Rab5a expression (Wainszelbaum et al., 2006). IL-4 stimulation of macrophages also results in prolonged retention of Rab5 on phagosomes and a delay in phagosome acidification in a phosphoinositide 3-kinase (PI3K)-dependent manner (Keijzer et al., 2011). Interestingly, IL-4/PGE2 enhance proteolytic activity in phagosomes (Balce et al., 2011). In contrast to IL-4 stimulation, overexpression of Rab5a in macrophages results in enhanced maturation of Listeria containing phagosomes (Alvarez-Dominguez and Stahl, 1999). The pro-inflammatory cytokine IL-6 similarly up-regulates Rab5 expression via activation of extracellular signal-regulated kinase (ERK) resulting in an increased fusion of early endosomes and phagosomes (Bhattacharya et al., 2006). Furthermore, the induction of Rab5a increases early endosome homotypic fusion thereby generating enlarged endosomal compartments (Wainszelbaum et al., 2006). The different effects in Rab5 expression after cytokine stimulation when compared with overexpression of Rab5 fusion proteins on phagosome maturation warrant further investigation. It also remains to be determined whether the observed differences in phagosomal acidification and proteolytic activity are linked to changes in Rab5a expression and endosomal morphology.

In contrast, expression of the late endosomal Rab7 is induced by IL-12 in a p38/MAPK-dependent manner (Bhattacharya et al., 2006). Increased Rab7 levels enhance transport of phagosomal cargo to lysosomes and inhibit the survival of intracellular Salmonella (Bhattacharya et al., 2006). In addition, the peptidoglycan constituent muramyl dipeptide, which is present in both gram positive and gram negative bacteria, has opposing effects on Rab5 and Rab7 levels in macrophages. Muramyl dipeptide decreases Rab5 levels while increasing Rab7 expression, corresponding with delivery of Salmonella to lysosomal compartments (Mukherjee et al., 2002).

Rab20 is an IFN-γ responsive Rab GTPase implicated in phagosome maturation and macropinocytosis (Egami and Araki, 2012a; Pei et al., 2014, 2015). IFN-γ was shown to up-regulate both total Rab20 levels (Pei et al., 2015) and its association with isolated bead-containing phagosomes (Trost et al., 2009). Further supporting the role of Rab20 in immunity, analysis of gene expression in lungs in the mouse model of asthma showed an increase of Rab20 expression after injection with ovalbumin (Malik et al., 2008). Furthermore, microarray analyses revealed Rab20 expression also increases in response to infections with pathogens including Aspergillus fumigatus (Cortez et al., 2006), Streptococcus pyogenes (Goldmann et al., 2007), and Listeria monocytogenes (Tchatalbachev et al., 2010). In addition, microarray data identified an increase in Rab20 levels during mycobacterial infection, which was dependent on NF-KB (Gutierrez et al., 2008). Simultaneous transcriptional profiling of M. tuberculosis and its infected host cells by microarrays showed a significant up-regulation of Rab20 in human macrophages but not in DCs (Tailleux et al., 2008). Remarkably, in a recent study of genes associated with the host transcriptional signature in active tuberculosis, Rab20 was the only small GTPase of the Rab family found in this set of 393 genes (Berry et al., 2010).

Supporting the idea that Rab20, together with Rab32, are part of a group of small GTPases linked to inflammation, the up-regulation of both Rab20 and Rab32 during the acute phase of LPS-induced brain inflammation has been reported (Liang et al., 2012). However, the mechanisms or consequences of this up-regulation are not known. Furthermore, high throughput siRNA screening to identify host pathways during L. monocytogenes infection in HeLa cells demonstrated that both Rab20 and Rab32 are required for the control of Listeria infection (Kuhbacher et al., 2015). While the direct transcriptional regulation of Rab32 in response to cytokines is unclear, some reports have directly linked Rab32 to immune responses in animal models of infection (Liang et al., 2012; Solano-Collado et al., 2016).

Given the critical roles of Rab proteins in immune related processes, they have been implicated in several immune disorders. Indeed, multiple genome-wide studies suggest that Rab protein-dependent cellular trafficking events are involved in immune responses (Pei et al., 2012). Disorders associated with dysfunctional Rab GTPase pathways can result from direct dysfunction of Rab proteins or indirectly as a result of defective Rab protein regulators or effectors, and can be genetic or occur during infection due to pathogen-driven processes (for reviews, see references Mitra et al., 2011; Seixas et al., 2013).

Several of the inherited Rab-associated disorders affect LROs and Rab27, Rab38, and Rab32 have been implicated in diseases with underlying defects in LRO trafficking. Interestingly, Rab27a is the only Rab GTPase that is clearly associated with an immune dysfunction in humans. Defects in Rab27a-dependent trafficking of melanosomes in melanocytes that affects the transfer of melanin to keratinocytes are associated with Griscelli syndrome, characterized by hair and skin hypopigmentation and impaired secretion of lytic granules from cytotoxic T lymphocytes, leading to immune-deficiencies (Menasche et al., 2000). Mutations in the Rab escort protein 1 (REP1), which is essential for prenylation of Rab GTPases, disrupt Rab27a trafficking through accumulation of unprenylated Rab27a, causing choroideremia (van den Hurk et al., 1997; Mitra et al., 2011). Moreover, defects in Rab27a-dependent transport of melanosomes in retinal-pigment epithelium is speculated to contribute to its degeneration leading to the loss of peripheral vision and night blindness associated with the disease (Mitra et al., 2011). Mutations in the Rab GTPase Rab38 also result in defective LRO trafficking and have been implicated in Hermansky–Pudlak syndrome in chocolate mice (Loftus et al., 2002). This hypopigmentation disorder is associated with impaired clotting due to the absence of platelet dense granules (Huizing et al., 2000). Rab38 and its close homolog Rab32 were also reported to control Salmonella and Listeria infection (Spano and Galan, 2012; Li et al., 2016). Furthermore, genetic evidence supports a role for Rab32 in controlling leprosy, caused by M. leprae (Zhang et al., 2011). While, the best-characterized function of Rab32 is its role in LRO trafficking, whether the Rab32-dependent LRO trafficking contributes to phagolysosome formation and pathogen clearance remains unknown.

Aberrant Rab protein expression is associated with diseases where chronic inflammation is speculated to contribute to disease causation and progression, including several types of cancers (Chia and Tang, 2009). Several hallmarks of cancer cells, such as altered cell polarity, require dysfunction of membrane trafficking events regulated by Rab GTPases. While the direct involvement of Rab proteins in cancer is not well established, abnormal expression of various Rab GTPases has been detected in several cancers (Chia and Tang, 2009; Goldenring, 2013), whereby aberrant Rab expression can be linked to varying phenotypes in different cancers. Rab25 and Rab21, which are involved in the trafficking of integrin receptors, are for example up-regulated in ovarian cancers and potentially promote cancer cell invasion (Cheng et al., 2004; Pellinen et al., 2006; Caswell et al., 2007). On the other hand, loss of Rab25 was associated with triple-negative breast cancer and head and neck cancers (Goldenring, 2013).

In addition to aberrant Rab expression, mistargeting of Rab GTPases or changes in their activity and localisation by posttranslational modifications is associated with various disorders. Chron's disease (CD) is a type of inflammatory bowel disease (IBD) characterized by a chronic inflammation of the gastrointestinal tract (Landy et al., 2016). This disease is associated with defects in cell-cell junctions resulting in loss of mucosal barrier integrity and increased permeability of the intestine (Teshima et al., 2012). Rab13 regulates the structure and function of tight junctions (Marzesco et al., 2002) and the mistargeting of Rab13 to basolateral sites observed in CD patients (Ohira et al., 2009) suggests a contribution of Rab13 dysfunction in CD. Several neurodegenerative disorders, including Parkinson's disease (PD), are associated with inflammation, where it is believed to cause neuronal degeneration and contribute to disease progression (Russo et al., 2014). Multiple studies support the role of leucine-rich repeat kinase 2 (LRRK2) in microglia-mediated inflammatory responses through regulation of vesicle trafficking, endocytosis and secretion (Russo et al., 2014). Interestingly, a recent phosphoproteomic screening revealed that several Rab GTPases act as LRRK2 substrates (Steger et al., 2016). This indicates a potential role for LRRK2-dependent Rab phosphorylation in regulating vesicle trafficking implicated in PD associated neuroinflammation.

In the last few years it has become evident that Rab GTPases, by regulating fundamental cellular processes, also regulate many important aspects of immune responses. Whereas some processes such as phagosome maturation and antigen presentation are well described, it is often not considered that secretion of cytokines relies on exocytosis; a membrane trafficking pathway regulated by Rab GTPases among other molecules. In addition, the expression of Rab GTPases is heavily regulated by cytokines, and there is a network of Rab proteins linked to various inflammatory processes that include Rab20 and Rab32. Future work will define the molecular basis of Rab gene expression during disease and their role during infection and immunity. In vivo studies with knock-out mice have demonstrated antimicrobial roles for Rab20 (Schnettger et al., 2017) and Rab32 (Solano-Collado et al., 2016), however roles of other Rab proteins using in vivo models remain largely undefined. Extending findings from in vitro studies to in vivo models will be crucial in understanding the roles of other Rab proteins in immune responses.

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.