Possible Multitasking of Stromal Cells in Lymphoid Organs – Hints for Glial Function

Multitasking stromal cells lymphoid organs glial function
Multitasking – stromal cells – lymphoid organs

Preamble in “multitasking”

In recent years, a new concept has emerged in many different areas of molecular cell biology, as many cells seem to do more than their well-known functions that have been partially described for decades. At the same time, it becomes more and more apparent, that totally different cell types use the same molecules – mainly proteins – for totally different tasks, often with marginal variations in protein structure. Some examples should be mentioned when applied to the nervous and immune systems.

In the immune system, myeloid cells like macrophages and microglia cells are described with non-immune functions. Macrophages may help to fortify the epithelial barrier, to maintain neuronal homeostasis in the enteric nervous system and to support the vascular system (Chiaranunt, Tai et al. 2021). Microglia help to remodel synapses and support myelination processes (Borst, Dumas et al. 2021). With regard to molecules with immune function, it is worth mentioning toll-like-receptors, which appear to have many more fields of application. Chen et al. (2019) found that these typical Pathogen recognition receptors are also expressed by neurons during developmental processes where they help the outgrowing neurites in finding their correct targets.

In the nervous system, only neurons seem to remain in their characteristic and highly specific niche of function as – to the authors’ knowledge – nothing is known about non-neuronal functions of these cells. On the other hand, beside the well-known function of nearly all glial cells in synaptic transmission and regulation, more and more non-neuronal cells are discovered with functions similar to neurons. As one example of such cells from totally different tissues, keratinocytes can detect thermal, mechanical and chemical stimuli and seem to also express neurotransmitter receptors (Mikesell, Isaeva et al. 2022, Xu, Yu et al. 2022) or Schwann-cells being involved in nociceptive transduction and regulation (Abdo, Calvo-Enrique et al. 2019) but also in classical immune functions (Meyer Zu Horste, Heidenreich et al. 2010). Last but not least, even voltage gated ion channels normally responsible for propagating action potentials along axons are expressed in non-neuronal cells fulfilling different functions. Macrophages as also keratinocytes use voltage gated sodium channels like Nav 1.8 for regulating RNA processing or for mediating inflammatory processes (White, Dungan et al. 2019, Zhang, Li et al. 2022).

The search for glial cells in lymphoid organs

Previous work done by our group has shown dense innervation patterns of single cells, so called “hard-wired” immune cells: wIC in murine and human lymph nodes (Wulfing and Gunther 2015). Later, we confirmed the presence of such cells in many other lymphoid organs and also discovered dense innervation patterns of probably free nerve endings always in antigen entrance sites like the floor of the lymph nodes subcapsular sinus or the subepithelial dome of Peyer’s patches, a phenomenon, we called neural nexus (Wulfing, Schuran et al. 2018). We also found many hints for an afferent character of this neural fibers, and now strongly suggest those innervation patterns to be part of a systematic neuro-immune communication being related to the proposed immunological homunculus (Wülfing et al. 2016 and 2019 on brainimmune.com).

However, as all axons are normally accompanied by glial cells whereas this task in the peripheral nervous system is done by Schwann cells, we wanted to know which glial cells accompany those axons as myelinating or non-myelinating Schwann cells (Gunther, Henne et al. 2021). Our suggestion was, that we have to search for non-myelinating Schwann cells, as axons and especially their end terminals of the autonomous nervous system are non-myelinated C-fibers. We also assumed a highly dynamic innervation of lymphoid organs so we decided to use GFAP (glial fibrillary acidic protein) as a marker. GFAP is widely used for marking astrocytes in the central nervous system, but beside this well-known function, many Schwann cells in the peripheral nervous system also express GFAP whenever there is an active and dynamic change in innervation patterns (Griffin and Thompson 2008).

To make a long story short, our results with a polyclonal GFAP antibody showed us immunohistochemical stainings, that resembled a classical silver staining of a lymph node (Figure 1) The whole reticular network seemed to be stained with GFAP, filamentary signals were present in the capsule, lining the subsinoidal layer, following sinus-structures, surrounding high endothelial venules and showing the typical reticular network in cortical, paracortical and medullary areas.

figure 1 T cell area lymph nodeFigure 1: (A) T-cell area of a lymph node. Immunohistochemical co-staining with anti-GFAP antibody (orange) and anti-neurofilament (green) – 20x. Beside the characteristic wIC signal stained by anti-neurofilament, a dense network of intermediate fibers is marked by anti-GFAP. Many signals seem to be in close relation to each other, but not co-localized. (B and C) Immunohistochemical staining with anti-GFAP antibody (red) and DAPI nuclear staining (blue). (B) capsule area with subcapsular sinus of a lymph node. Note the localization of the filamentous GFAP signal exactly throughout the sinus, it seems to be closer to the floor of the capsular sinus – 40x. (C) T-cell area close to a follicle in a lymph node. Note the GFAP signal surrounding the follicle (arrows) but also surrounding the high endothelial venules (asterix) – 20x. GFAP: glial fibrillary acidic protein, wIC: hard wired immune cell.

As we suspected, we detected other types of cells and not only Schwann cells in that amount, so we also did stainings with desmin, a typical intermediate filament of stromal cells. Interestingly, we experienced an expressive but not complete co-localization between GFAP and desmin, which are different intermediate filaments (Figure 2).

figure 2 T cell area lymph nodeFigure 2: T-cell area of a lymph node. Immunohistochemical co-staining with anti-GFAP antibody (red), anti-desmin antibody (green) and DAPI nuclear staining (blue) – 20x. Note the dense network of filamentous signals resembling a reticular network similar observed with a classical silver staining. The signals for GFAP and desmin seem to co-localize at many parts, but not completely. GFAP: glial fibrillary acidic protein.

Other stainings for stromal cells like LYVE-1 (lymphatic endothelial cells), CD 31 (blood endothelial cells) and podoplanin (fibroblastic reticular cells) confirmed that stromal cells were marked by both GFAP and desmin antibodies (Figure 3).

To complete the picture, we used a monoclonal antibody against GFAP and performed FISH analysis. The results marked cells only in the capsule, hilus, and medulla of the lymph node, excluding the cortical and paracortical areas. These were precisely the microdomains where we had not found the dense innervation of immune cells and the neural nexus.

So together with the fact that we have detected other typical Schwann-cell markers like Myelin basic protein or S100 also only in medullary and hilus areas, one should conclude that Schwann cells are only present in the entrance areas of the axons into the lymph node.

figure 3 T cell area lymph nodeFigure 3: T-cell area of a lymph node. Immunohistochemical co-staining with anti-GFAP antibody (red), anti-podoplanin antibody (white) and anti-collagen III antibody (green) – 40x. The horizontal green line marks the orthographic section on the top (green frame), the vertical red line marks the orthographic section to the right (red frame). Note the close association between all the signals that seem to be related to the conduit system. The orthographic sections reveal that the podoplanin signal is located in between the collagen III and the GFAP signal (sequence: red-white-green), see blue circles. GFAP: glial fibrillary acidic protein.

But then the question remains open which cells accompany the axon terminals inside the lymph node parenchyma through paracortical T-cell areas and into follicular B-cell regions and their interfollicular regions up to the subcapsular sinus?

The concept of lymphoid organ stromal cells multitasking in glial functions

In recent years, not only have more and more non-neuronal cells been discovered that seem to fulfill typical neuronal functions like those mentioned above, but glial functions of these cells have also been identified. Especially epithelial cells like corneal epithelial cells or keratinocytes of the epidermis seem to surrogate Schwann cell functions (Stepp, Tadvalkar et al. 2017, Lowy, Makker et al. 2021).

Looking at lymphoid organs, most of the many diverse stromal cells have epithelial character. Certainly, one would classify all lymphatic endothelial cells lining the subcapsular sinus, the lymphatic labyrinths or the medullary sinuses as cells with epithelial character. Of course, the same epithelial character is defined for all blood endothelial cells coursing through the lymphoid organ parenchyma. But with uncovering the conduit system in lymph nodes, most of the stromal cells contributing to the formation of the conduits like fibroblastic reticular cells or marginal reticular cells also show epithelial characteristics: For example, the conduit system is defined as the spatial continuation of the extracellular space like the lymphatics. And the fibroblastic reticular cells forming the conduit are also producing a basement membrane like epithelial cells do, and this basement membrane is the inner lining of the channel-like tubular conduit system (Roozendaal, Mebius et al. 2008, Fletcher, Acton et al. 2015).

Summarizing the knowledge about cells fulfilling more than one classical function with one impressive example of epithelial cells being able to take over neuronal or glial function the idea of a new concept arose:

“Do stromal cells in lymphoid organs also have glial functions?”

Of course, such a concept will need a lot of further investigations, especially functional approaches. Nevertheless, for now, we decided to focus on other possible glial characteristics of stromal cells, so we did more co-stainings with other markers. Connexin 43 is described as a gap junction protein often expressed by astrocytes like GFAP (Boulay, Cisternino et al. 2016). Interestingly, we also found a close association between a Connexin 43 and the GFAP signal which can be seen in (Figures 4 and Figure 5).

figure 4 T-cell area lymph nodeFigure 4: T-cell area of a lymph node. Immunohistochemical co-staining with anti-GFAP antibody (red), anti-connexin 43 antibody (orange) and DAPI nuclear staining (blue) – 40x. The horizontal green line marks the orthographic section on the top (green frame), the vertical red line marks the orthographic section to the right (red frame). Note the close association between the “dotted” connexin 43 signal revealing the morphology of a cell and the close association to the filamentous GFAP signal. The orthographic sections confirm the close spatial association, see blue circles. GFAP: glial fibrillary acidic protein.

Most striking was the near association of the connexin 43 signal with the GFAP and neurofilament signal which can be observed in the orthogonal view in (Figure 5).

But that’s not all, a marker for a glial cell normally only present in the central nervous system showed positive results: for oligodendrocytes (Figure 6). We later confirmed the presence of oligodendrocyte like cells myelinating more than one axon in the medulla of lymph nodes via electron microscopy (not shown, already published – (Gunther, Henne et al. 2021).

figure 5 T-cell area lymph nodeFigure 5: T-cell area of a lymph node. Immunohistochemical co-staining with anti-GFAP antibody (red), anti-connexin 43 antibody (orange), anti-neurofilament antibody (green) and DAPI nuclear staining (blue) – 40x. The horizontal green line marks the orthographic section on the top (green frame), the vertical red line marks the orthographic section to the right (red frame). One can observe the impressive co-localization of neuronal and glial signals, again resembling the wIC structures already described by us in former work. Especially the orthographic view to the right reveals that the connexin 43 signal is located in between the GFAP signal and the neurofilament signal (sequence: red-orange-green), see blue circle. GFAP: glial fibrillary acidic protein, wIC: hard wired immune cell.

We therefore suggest stromal cells fulfill glial functions as one additional role and would like to speculate about the possibility, that stromal cells in lymph nodes therefore express different intermediate filament types, dependent on the necessary function in each part of the cell. It is well known that cells can express more than one type of intermediate filament, even different classes of intermediate filaments in the same cell. It is also known that GFAP and desmin both belong to class III, as they have a strong sequence homology. Moreover, the functional role of intermediate filaments is mostly determined by the tail region and its phosphorylation sites. For an excellent review see (Bott and Winckler 2020).

figure 6 Medullary area lymph nodeFigure 6: Medullary area of a lymph node. Immunohistochemical co-staining with anti-GFAP antibody (red), anti-oligodendrocyte antibody (orange) and DAPI nuclear staining (blue) – 40x. (A) overview of the medullary area showing the orange oligodendrocyte signal clearly separated from the GFAP signal – 20x. (B) Closer view of the blue frame in A that confirmed both signals to be associated but not overlapping. – 40x. GFAP: glial fibrillary acidic protein.

As different phosphorylation of the tail region will change the spatial configuration in the close vicinity of the intermediate filaments, it will probably also change the possible binding sites for antibodies. And this may be the reason for the inhomogeneous results with monoclonal and polyclonal GFAP antibodies in relation to the results with desmin antibodies. In our proposed model, the stromal cell in lymph nodes will use different intermediate filament types with different tail phosphorylation states in different parts of the cell dependent on the functions necessary in that region. We hypothesize this illustration for one type of stromal cell, the fibroblastic reticular cell (FRC), that forms the conduit system throughout the whole organ, this type of stromal cell would be able to accompany axons throughout their complete course (Figure 7).

figure 7 Schematic drawing conceptFigure 7: Schematic drawing of the concept proposed in this short article. Two fibroblastic reticular cells (FRC – stromal cells of the lymph node) as an example for stromal cells can be seen, forming a conduit in between with their cytoplasmatic extensions (cells surrounded by the plasma membrane, conduit surrounded by basement membrane). The conduit itself is the continued extracellular space, therefore “filled” by collagen fibers type III and type I. All markers identified by us are shown only with the cell on the bottom right. Beside the typical FRC surface marker podoplanin, the FRC produce a basement membrane where they face the extracellular space of the conduit, giving them epithelial character. At the subcellular locations where the cytoskeleton needs to connect to cell adhesions like desmosomes or hemidesmosomes the FRC may express desmin as their intermediate filament for structural purposes. At other subcellular locations where the FRC may accompany axons (2 round circles at the bottom filled by neurofilament) they may express GFAP fulfilling probably glial functions in our model proposed. GFAP: glial fibrillary acidic protein, FRC: fibroblastic reticular cell.

An expressive example underlining our suggested model can be seen in (Figure 8), where the neurofilament signal reaching one single wIC is closely associated to the GFAP signal which in turn surrounds a tubular structure marked with a collagen III signal inside.

Of course, we also have to bear in mind that all what we see is simply based on a non-specific binding and false positive results of the different antibodies. But the additional results with other glial markers as the morphology of the structures we see in close association to each other strongly supports our suggested glial function of stromal cells in lymphoid organs. Consequently, for now we would like to propose a model of a multitasking stromal cell that also fulfills glial functions in lymph nodes. If this can be confirmed with further work, it is also very probable that all stromal cells are part of the suggested complex and systematic neuro-immune communication explained at the beginning of this article.

figure 8 T-cell area lymph nodeFigure 8: T-cell area of a lymph node. Immunohistochemical co-staining with anti-GFAP antibody (orange), anti-collagen III antibody (red) and anti-neurofilament antibody (green) – 40x. (A) The overview shows the typical conduit system marked by the red collagen III signal. In the white frame, the conduit seems to be enclosed by the orange GFAP signal and a cell which is marked by the green neurofilament signal is located close by. (B) 3D Z-stack of the white frame in A which more clearly shows the close association between the two filamentous signals of neurofilament and GFAP (white arrows).  GFAP: glial fibrillary acidic protein.

Abdo, H., et al. (2019). “Specialized cutaneous Schwann cells initiate pain sensation.” Science 365(6454): 695-699.

An essential prerequisite for the survival of an organism is the ability to detect and respond to aversive stimuli. Current belief is that noxious stimuli directly activate nociceptive sensory nerve endings in the skin. We discovered a specialized cutaneous glial cell type with extensive processes forming a mesh-like network in the subepidermal border of the skin that conveys noxious thermal and mechanical sensitivity. We demonstrate a direct excitatory functional connection to sensory neurons and provide evidence of a previously unknown organ that has an essential physiological role in sensing noxious stimuli. Thus, these glial cells, which are intimately associated with unmyelinated nociceptive nerves, are inherently mechanosensitive and transmit nociceptive information to the nerve.

Borst, K., et al. (2021). “Microglia: Immune and non-immune functions.” Immunity 54(10): 2194-2208.

As resident macrophages of the central nervous system (CNS), microglia are associated with diverse functions essential to the developing and adult brain during homeostasis and disease. They are aided in their tasks by intricate bidirectional communication with other brain cells under steady-state conditions as well as with infiltrating peripheral immune cells during perturbations. Harmonious cell-cell communication involving microglia are considered crucial to maintain the healthy state of the tissue environment and to overcome pathology such as neuroinflammation. Analyses of such intercellular pathways have contributed to our understanding of the heterogeneous but context-associated microglial responses to environmental cues across neuropathology, including inflammatory conditions such as infections and autoimmunity, as well as immunosuppressive states as seen in brain tumors. Here, we summarize the latest evidence demonstrating how these interactions drive microglia immune and non-immune functions, which coordinate the transition from homeostatic to disease-related cellular states.

Bott, C. J. and B. Winckler (2020). “Intermediate filaments in developing neurons: Beyond structure.” Cytoskeleton (Hoboken) 77(3-4): 110-128.

Neuronal development relies on a highly choreographed progression of dynamic cellular processes by which newborn neurons migrate, extend axons and dendrites, innervate their targets, and make functional synapses. Many of these dynamic processes require coordinated changes in morphology, powered by the cell’s cytoskeleton. Intermediate filaments (IFs) are the third major cytoskeletal elements in vertebrate cells, but are rarely considered when it comes to understanding axon and dendrite growth, pathfinding and synapse formation. In this review, we first introduce the many new and exciting concepts of IF function, discovered mostly in non-neuronal cells. These roles include dynamic rearrangements, crosstalk with microtubules and actin filaments, mechano-sensing and -transduction, and regulation of signaling cascades. We then discuss the understudied roles of neuronally expressed IFs, with a particular focus on IFs expressed during development, such as nestin, vimentin and alpha-internexin. Lastly, we illustrate how signaling modulation by the unconventional IF nestin shapes neuronal morphogenesis in unexpected and novel ways. Even though the first IF knockout mice were made over 20 years ago, the study of the cell biological functions of IFs in the brain still has much room for exciting new discoveries.

Boulay, A. C., et al. (2016). “Immunoregulation at the gliovascular unit in the healthy brain: A focus on Connexin 43.” Brain Behav Immun 56: 1-9.

In the brain, immune cell infiltration is normally kept at a very low level and a unique microenvironment strictly restricts immune reactions and inflammation. Even in such quiescent environment, a constant immune surveillance is at work allowing the brain to rapidly react to threats. To date, knowledge about the factors regulating the brain-immune system interrelationship in healthy conditions remains elusive. Interestingly, astrocytes, the most abundant glial cells in the brain, may participate in many aspects of this unique homeostasis, in particular due to their close interaction with the brain vascular system and expression of a specific molecular repertoire. Indeed, astrocytes maintain the blood-brain barrier (BBB) integrity, interact with immune cells, and participate in the regulation of intracerebral liquid movements. We recently showed that Connexin 43 (Cx43), a gap junction protein highly expressed by astrocytes at the BBB interface, is an immunoregulating factor. The absence of astroglial Cx43 leads to a transient endothelial activation, a continuous immune recruitment as well as the development of a specific humoral autoimmune response against the von Willebrand factor A domain-containing protein 5a, an extracellular matrix protein expressed by astrocytes. In this review, we propose to gather current knowledge on how astrocytes may influence the immune system in the healthy brain, focusing on their roles at the gliovascular interface. We will also consider pathological situations involving astrocyte-specific autoimmunities. Finally, we will discuss the specific role of astroglial Cx43 and the physiological consequences of immune regulations taking place on inflammation, cognition and behavior in the absence of Cx43.

Chen, C. Y., et al. (2019). “Beyond defense: regulation of neuronal morphogenesis and brain functions via Toll-like receptors.” J Biomed Sci 26(1): 90.

Toll-like receptors (TLRs) are well known as critical pattern recognition receptors that trigger innate immune responses. In addition, TLRs are expressed in neurons and may act as the gears in the neuronal detection/alarm system for making good connections. As neuronal differentiation and circuit formation take place along with programmed cell death, neurons face the challenge of connecting with appropriate targets while avoiding dying or dead neurons. Activation of neuronal TLR3, TLR7 and TLR8 with nucleic acids negatively modulates neurite outgrowth and alters synapse formation in a cell-autonomous manner. It consequently influences neural connectivity and brain function and leads to deficits related to neuropsychiatric disorders. Importantly, neuronal TLR activation does not simply duplicate the downstream signal pathways and effectors of classical innate immune responses. The differences in spatial and temporal expression of TLRs and their ligands likely account for the diverse signaling pathways of neuronal TLRs. In conclusion, the accumulated evidence strengthens the idea that the innate immune system of neurons serves as an alarm system that responds to exogenous pathogens as well as intrinsic danger signals and fine-tune developmental processes of neurons.

Chiaranunt, P., et al. (2021). “Beyond Immunity: Underappreciated Functions of Intestinal Macrophages.” Front Immunol 12: 749708.

The gastrointestinal tract hosts the largest compartment of macrophages in the body, where they serve as mediators of host defense and immunity. Seeded in the complex tissue-environment of the gut, an array of both hematopoietic and non-hematopoietic cells forms their immediate neighborhood. Emerging data demonstrate that the functional diversity of intestinal macrophages reaches beyond classical immunity and includes underappreciated non-immune functions. In this review, we discuss recent advances in research on intestinal macrophage heterogeneity, with a particular focus on how non-immune functions of macrophages impact tissue homeostasis and function. We delve into the strategic localization of distinct gut macrophage populations, describe the potential factors that regulate their identity and functional heterogeneity within these locations, and provide open questions that we hope will inspire research dedicated to elucidating a holistic view on macrophage-tissue cell interactions in the body’s largest mucosal organ.

Fletcher, A. L., et al. (2015). “Lymph node fibroblastic reticular cells in health and disease.” Nat Rev Immunol 15(6): 350-361.

Over the past decade, a series of discoveries relating to fibroblastic reticular cells (FRCs) – immunologically specialized myofibroblasts found in lymphoid tissue – has promoted these cells from benign bystanders to major players in the immune response. In this Review, we focus on recent advances regarding the immunobiology of lymph node-derived FRCs, presenting an updated view of crucial checkpoints during their development and their dynamic control of lymph node expansion and contraction during infection. We highlight the robust effects of FRCs on systemic B cell and T cell responses, and we present an emerging view of FRCs as drivers of pathology following acute and chronic viral infections. Lastly, we review emerging therapeutic advances that harness the immunoregulatory properties of FRCs.

Griffin, J. W. and W. J. Thompson (2008). “Biology and pathology of nonmyelinating Schwann cells.” Glia 56(14): 1518-1531.

The CNS contains relatively few unmyelinated nerve fibers, and thus benefits from the advantages that are conferred by myelination, including faster conduction velocities, lower energy consumption for impulse transmission, and greater stability of point-to-point connectivity. In the PNS many fibers or regions of fibers the Schwann do not form myelin. Examples include C fibers nociceptors, postganglionic sympathetic fibers, and the Schwann cells associated with motor nerve terminals at neuromuscular junctions. These examples retain a degree of plasticity and a capacity to sprout collaterally that is unusual in myelinated fibers. Nonmyelin-forming Schwann cells, including those associated with uninjured fibers, have the capacity to act as the “first responders” to injury or disease in their neighborhoods.

Gunther, H. S., et al. (2021). “GFAP and desmin expression in lymphatic tissues leads to difficulties in distinguishing between glial and stromal cells.” Sci Rep 11(1): 13322.

Recently, we found many immune cells including antigen presenting cells neurally hard wired in the T-cell zone of most lymphoid organs like amongst others, lymph nodes in rats, mice and humans. Single immune cells were reached by single neurites and enclosed with a dense neural meshwork. As it is well known that axons are always accompanied by glial cells, we were able to identify Schwann cells in the hilum, medullary and capsule region, like expected. Unexpected was the result, that we found oligodendrocyte-like cells in these regions, myelinating more than one axon. Likewise important was the finding, that one of the standard glial markers used, a polyclonal GFAP antibody equally bound to desmin and therefore marked nearly all stromal cells in cortical, paracortical and medullary cord regions. More detailed analysis showed that these results also appeared in many other non-lymphoid organs. Therefore, polyclonal GFAP antibodies are only conditionally usable for immunohistochemical analysis in peripheral tissues outside the central nervous system. It remains to be elucidated, if the binding of the GFAP antibody to desmin has its reason in a special desmin variant that can give stromal cells glial character.

Lowy, D. B., et al. (2021). “Cutaneous Neuroimmune Interactions in Peripheral Neuropathic Pain States.” Front Immunol 12: 660203.

Bidirectional interplay between the peripheral immune and nervous systems plays a crucial role in maintaining homeostasis and responding to noxious stimuli. This crosstalk is facilitated by a variety of cytokines, inflammatory mediators and neuropeptides. Dysregulation of this delicate physiological balance is implicated in the pathological mechanisms of various skin disorders and peripheral neuropathies. The skin is a highly complex biological structure within which peripheral sensory nerve terminals and immune cells colocalise. Herein, we provide an overview of the sensory innervation of the skin and immune cells resident to the skin. We discuss modulation of cutaneous immune response by sensory neurons and their mediators (e.g., nociceptor-derived neuropeptides), and sensory neuron regulation by cutaneous immune cells (e.g., nociceptor sensitization by immune-derived mediators). In particular, we discuss recent findings concerning neuroimmune communication in skin infections, psoriasis, allergic contact dermatitis and atopic dermatitis. We then summarize evidence of neuroimmune mechanisms in the skin in the context of peripheral neuropathic pain states, including chemotherapy-induced peripheral neuropathy, diabetic polyneuropathy, post-herpetic neuralgia, HIV-induced neuropathy, as well as entrapment and traumatic neuropathies. Finally, we highlight the future promise of emerging therapies associated with skin neuroimmune crosstalk in neuropathic pain.

Meyer Zu Horste, G., et al. (2010). “Expression of antigen processing and presenting molecules by Schwann cells in inflammatory neuropathies.” Glia 58(1): 80-92.

Schwann cells are the myelinating glia cells of the peripheral nervous system (PNS) and can become targets of an autoimmune response in inflammatory neuropathies like the Guillain-Barre syndrome (GBS). Professional antigen presenting cells (APCs) are known to promote autoimmune responses in target tissues by presenting self-antigens. Other cell types could participate in local autoimmune responses by acting as nonprofessional APCs. Using a combined approach of immunocytochemistry, immunohistochemistry, and flow cytometry analysis we demonstrate that human Schwann cells express the antigen processing and presenting machinery (APM) in vitro and in vivo. Moreover, cultured human Schwann cells increase the expression of proteasome subunit delta (Y), antigen peptide transporter TAP2, and HLA Class I and HLA Class II complexes in an inflammatory environment. In correlation with this observation, Schwann cells in sural nerve biopsies from GBS patients show increased expression of antigen processing and presenting molecules. Furthermore, cultured human Schwann cells can proteolytically digest fluorescently-labeled nonmammalian antigen ovalbumin. Taken together, our data suggest antigen processing and presentation as a possible function of Schwann cells that may contribute to (auto)immune responses within peripheral nerves.

Mikesell, A. R., et al. (2022). “Keratinocyte PIEZO1 modulates cutaneous mechanosensation.” Elife 11.

Epidermal keratinocytes mediate touch sensation by detecting and encoding tactile information to sensory neurons. However, the specific mechanotransducers that enable keratinocytes to respond to mechanical stimulation are unknown. Here, we found that the mechanically-gated ion channel PIEZO1 is a key keratinocyte mechanotransducer. Keratinocyte expression of PIEZO1 is critical for normal sensory afferent firing and behavioral responses to mechanical stimuli in mice.

Roozendaal, R., et al. (2008). “The conduit system of the lymph node.” Int Immunol 20(12): 1483-1487.

The lymphoid compartment of lymph nodes is impermeable to many molecules that are delivered via afferent lymphatic vessels. In the lymphoid compartment, fibroblast reticular cells form an interconnected network-the conduit system. This network has a structural function supporting tightly packed lymphocytes and antigen-presenting cells; however, it also has an important function as a molecular sieve, since it contains tubules that are the only entry point for fluid and allow only small molecules and particles (including antigens) to flow along the network. This size exclusion may prevent pathogens entering the blood from lymph. Dendritic cells can sample antigens from the conduit system and present them to nearby lymphocytes; this may be particularly important in initiating immune responses. The importance of larger antigen transport via macrophages or other cells is unclear. Lymphocytes and antigen-presenting dendritic cells actively move and interact along the conduit system, perhaps in response to chemokines or cytokines transported by the conduit system; these molecules may also be transported to high endothelial venules and regulate the attraction of blood leukocytes to the lymph nodes. The conduit system is also important for fluid distribution between afferent lymphatics and blood, but the mechanisms are not yet established.

Stepp, M. A., et al. (2017). “Corneal epithelial cells function as surrogate Schwann cells for their sensory nerves.” Glia 65(6): 851-863.

The eye is innervated by neurons derived from both the central nervous system and peripheral nervous system (PNS). While much is known about retinal neurobiology and phototransduction, less attention has been paid to the innervation of the eye by the PNS and the roles it plays in maintaining a functioning visual system. The ophthalmic branch of the trigeminal ganglion contains somas of neurons that innervate the cornea. These nerves provide sensory functions for the cornea and are referred to as intraepithelial corneal nerves (ICNs) consisting of subbasal nerves and their associated intraepithelial nerve terminals. ICNs project for several millimeters within the corneal epithelium without Schwann cell support. Here, we present evidence for the hypothesis that corneal epithelial cells function as glial cells to support the ICNs. Much of the data supporting this hypothesis is derived from studies of corneal development and the reinnervation of the ICNs in the rodent and rabbit cornea after superficial wounds. Corneal epithelial cells activate in response to injury via mechanisms similar to those induced in Schwann cells during Wallerian Degeneration. Corneal epithelial cells phagocytize distal axon fragments within hours of ICN crush wounds. During aging, the proteins, lipids, and mitochondria within the ICNs become damaged in a process exacerbated by UV light. We propose that ICNs shed their aged and damaged termini and continuously elongate to maintain their density. Available evidence points to new unexpected roles for corneal epithelial cells functioning as surrogate Schwann cells for the ICNs during homeostasis and in response to injury. GLIA 2017;65:851-863.

White, C. R., et al. (2019). “Activation of human macrophage sodium channels regulates RNA processing to increase expression of the DNA repair protein PPP1R10.” Immunobiology 224(1): 80-93.

Prior work demonstrated that a splice variant of SCN5A, a voltage-gated sodium channel gene, acts as a cytoplasmic sensor for viral dsRNA in human macrophages. Expression of this channel also polarizes macrophages to an anti-inflammatory phenotype in vitro and in vivo. Here we utilized global expression analysis of splice variants to identify novel channel-dependent signaling mechanisms. Pharmacological activation of voltage-gated sodium channels in human macrophages, but not treatment with cytoplasmic poly I:C, was associated with splicing of a retained intron in transcripts of PPP1R10, a regulator of phosphatase activity and DNA repair. Microarray analysis also demonstrated expression of a novel sodium channel splice variant, human macrophage SCN10A, that contains a similar exon deletion as SCN5A. SCN10A localizes to cytoplasmic and nuclear vesicles in human macrophages. Simultaneous expression of human macrophage SCN5A and SCN10A was required to decrease expression of the retained intron and increase protein expression of PPP1R10. Channel activation also increased protein expression of the splicing factor EFTUD2, and knockdown of EFTUD2 prevented channel dependent splicing of the retained PPP1R10 intron. Knockdown of the SCN5A and SCN10A variants in human macrophages reduced the severity of dsDNA breaks induced by treatment with bleomycin and type 1 interferon. These results suggested that human macrophage SCN5A and SCN10A variants mediate an innate immune signaling pathway that limits DNA damage through increased expression of PPP1R10. The functional significance of this pathway is that it may prevent cytotoxicity during inflammatory responses.

Wulfing, C. and H. S. Gunther (2015). “Dendritic cells and macrophages neurally hard-wired in the lymph node.” Sci Rep 5: 16866.

The neural hard-wired pathways in which the lymphoid organs are innervated by the nervous system is of special interest with respect to suggested afferent and sensory systems informing the central nervous system about the status of the immune system. Until today efferent also like afferent innervation seem to be unspecific, targeting many types of cells by affecting many cells at the same time. We for the first time show that antigen presenting cells (APC) are abundantly innervated in the T-cell enriched area, the subsinoidal layer and the cortical extrafollicular zone of lymph nodes in rats by a mesh of filamentous neurofilament positive structures originating from single nerve fibers and covering each single APC similar to a glass fishing float, so that we termed them “wired” APC (wAPC). These wAPC also found in humans seem to be restricted to the cell body, not to follow membranous extensions, they may be dynamic and receptive as MAP2 is expressed and axonal growth cones can be detected and they probably lack vesicular activity through missing synaptophysin expression. The specific innervation targeting single cells which show a distribution divided in several areas in one lymph node suggests a form of topographically organized afferent sensory system.

Wulfing, C., et al. (2018). “Neural architecture in lymphoid organs: Hard-wired antigen presenting cells and neurite networks in antigen entrance areas.” Immun Inflamm Dis 6(2): 354-370.

INTRODUCTION: Recently, we found abundant innervation of antigen presenting cells that were reached and enclosed by single neurites. These neurally hard-wired antigen presenting cells (wAPC) could be observed in the T-cell zone of superficial cervical lymph nodes of rats and other mammalians, including humans. METHODS: As a consequence, we investigated lymph nodes at many different anatomical positions as well as all primary and secondary lymphoid organs (SLO) in rodents for a similar morphology of innervation regarding antigen presenting cells known in those tissues. RESULTS: As a result, we confirmed wAPC in lymph nodes independent from their draining areas and anatomical positions but also in all other T-cell zones of lymphoid organs, like Peyer’s patches, NALT and BALT, as well as in the thymic medulla. Other cells were innervated in a similar fashion but with seemingly missing antigen presenting capacity. Both types of innervated immune cells were observed as being also present in the dermis of the skin. Only in the spleen wAPC could not be detected. Beyond this systematic finding, we also found another regular phenomenon: a dense network of neurites that stained for neurofilament always in antigen entrance areas of lymphoid organs (subsinoidal layer of lymph nodes, subepithelial dome of Peyer’s patches, subsinoidal layer of the splenic white pulp, margins of NALT and BALT). Lastly, also thymic epithelial cells (TEC) restricted to the corticomedullary junction of the thymus showed similar neurofilament staining. CONCLUSIONS: Therefore, we propose much more hard-wired and probably afferent connections between lymphoid organs and the central nervous system than is hitherto known.

Xu, X., et al. (2022). “Emerging roles of keratinocytes in nociceptive transduction and regulation.” Front Mol Neurosci 15: 982202.

Keratinocytes are the predominant block-building cells in the epidermis. Emerging evidence has elucidated the roles of keratinocytes in a wide range of pathophysiological processes including cutaneous nociception, pruritus, and inflammation. Intraepidermal free nerve endings are entirely enwrapped within the gutters of keratinocyte cytoplasm and form en passant synaptic-like contacts with keratinocytes. Keratinocytes can detect thermal, mechanical, and chemical stimuli through transient receptor potential ion channels and other sensory receptors. The activated keratinocytes elicit calcium influx and release ATP, which binds to P2 receptors on free nerve endings and excites sensory neurons. This process is modulated by the endogenous opioid system and endothelin. Keratinocytes also express neurotransmitter receptors of adrenaline, acetylcholine, glutamate, and gamma-aminobutyric acid, which are involved in regulating the activation and migration, of keratinocytes. Furthermore, keratinocytes serve as both sources and targets of neurotrophic factors, pro-inflammatory cytokines, and neuropeptides. The autocrine and/or paracrine mechanisms of these mediators create a bidirectional feedback loop that amplifies neuroinflammation and contributes to peripheral sensitization.

Zhang, Y., et al. (2022). “Nav1.8 in keratinocytes contributes to ROS-mediated inflammation in inflammatory skin diseases.” Redox Biol 55: 102427.

Reactive oxygen species (ROS)-activated proinflammatory signals in keratinocytes play a crucial role in the immunoregulation of inflammatory skin diseases, including rosacea and psoriasis. Nav1.8 is a voltage-gated sodium ion channel, and its abnormal expression in the epidermal layer contributes to pain hypersensitivity in the skin. However, whether and how epidermal Nav1.8 is involved in skin immunoregulation remains unclear. This study was performed to identify the therapeutic role of Nav1.8 in inflammatory skin disorders. We found that Nav1.8 expression was significantly upregulated in the epidermis of rosacea and psoriasis skin lesions. Nav1.8 knockdown ameliorated skin inflammation in LL37-and imiquimod-induced inflammation mouse models. Transcriptome sequencing results indicated that Nav1.8 regulated the expression of pro-inflammatory mediators (IL1beta and IL6) in keratinocytes, thereby contributing to immune infiltration in inflammatory skin disorders. In vitro, tumor necrosis factor alpha (TNFalpha), a cytokine that drives the development of various inflammatory skin disorders, increased Nav1.8 expression in keratinocytes. Knockdown of Nav1.8 eliminated excess ROS production, thereby attenuating the TNFalpha-induced production of inflammatory mediators; however, a Nav1.8 blocker did not have the same effect. Mechanistically, Nav1.8 reduced superoxide dismutase 2 (SOD2) activity by directly binding to SOD2 to prevent its deacetylation and mitochondrial localization, subsequently inducing ROS accumulation. Collectively, our study describes a central role for Nav1.8 in regulating pro-inflammatory responses in the skin and indicates a novel therapeutic strategy for rosacea and psoriasis.