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Pain Sensitivity, NGF and How Dermal Macrophages Pull the Strings

In cases where the skin suffers a damage, it’s not just the sensory pain neural pathway that get into action. The skin also releases inflammatory mediators, often from activated nociceptors or non-neural cells such as macrophages and mast cells that are either residents or unexpected guests at the site of injury. Particularly interesting are skin-associated macrophages, which not only help in nerve regeneration but also play roles in modulating pain under certain conditions. Work published in Nature Immunology has shown how secretion of nerve growth factor (NGF) by dermal macrophages significantly influences how pain is experienced.

NGF and SNX25: Old Meets New

NGF is a secreted protein and a member of the neurotrophic family of growth factors. Remarkably, mutations in the Ngf gene in humans result in a specific neuropathy known as HSAN V, where affected individuals exhibit a significant absence of pain sensibility.

In th recent paper from Nature Immunology mentioned above, a serendipitous discovery of a pain-insensitive transgenic mouse line has shed light on the involvement of the Snx25 gene in pain modulation. Known for their roles in membrane trafficking and cell signaling, SNX proteins are now being shown to have a multitude of cellular functions. In particular, the expression of SNX25 in dermal macrophages has been shown to induces the production and secretion of NGF, thus modulating acute pain perception under both normal and pathological conditions.

What’s fascinating about this new research is the mechanism of SNX25 action: SNX25 induces the production of NGF by inhibiting the ubiquitin-mediated degradation of Nrf2, a transcription factor that kickstarts Ngf mRNA transcription. This signaling cascade is part of a complex interaction between macrophages and neurons that has broad implications for pain processing, both in healthy skin and under conditions like neuropathy or inflammation.

While the complex biological mechanism of skin and its interactions with peripheral sensory neurons and immune cells such as macrophages is beginning to be revealed, discoveries like these provide critical insights. Here we take a look at this new research that contributes to our understanding of sensory biology and begins to point us toward new avenues for therapeutic interventions in chronic and acute pain conditions.

The Unexpected Phenotype: Diminished Pain Responses in Snx25+/− Mice

Interestingly, Snx25+/− mice came about serendipitously through experiments involving the Mlc1 gene, which is associated with a congenital leukoencephalopathy. DNA sequencing revealed that the insertion of Mlc1 gene in mice led to the deletion of three genes – one of them was Snx25.

The team designed further experiments to assess SNX25 role in pain sensation. Snx25-knockout (KO) mice were created, and behavioral tests revealed similar patterns of reduced pain sensitivity, akin to Mlc1 transgenic mice.

The behavioral pain test used in this work was von Frey’s (VF) filaments method. In this test monofilaments are applied at increasing forces to the animal hind paw, and the threshold for animal response is determined.

Figure 1. Monofilaments of differing forces are applied perpendicularly to the hind paw. If the rodent withdraws, licks or shakes the paw, it is considered to have had a positive response. Deuis et al, Front Mol Neurosci 10: 284 (2017)

What the Neurons Say

Could this reduced sensitivity to pain result from abnormal neuron development or structure? Surprisingly, the cellular size distribution and expression of markers in small and large sensory neurons in the DRG showed no notable differences between adult Snx25+/− mice and their wild-type (WT) counterparts. Furthermore, this pain-insensitive trait in Snx25+/− mice became evident only after three weeks of age, ruling out developmental issues as the underlying cause.

By reducing SNX25 expression specifically in DRG neurons, the research shows that pain sensitivity remains intact, implying that the role of SNX25 in pain sensitivity is not associated with its expression at the DRG tissue.

Macrophage SNX25 and Pain Modulation

The exploration didn’t stop there; the research specifically targeted Snx25 in monocytes and macrophages to understand its role in pain regulation. Mice with a conditional deletion of Snx25 in these cells displayed heightened VF thresholds and reduced response to the painful formalin injection, suggesting insensitivity to pain. Moreover, this effect correlated with a significant decrease in the expression of pain-associated genes and chemokines, indicating that SNX25 in macrophages not only contributes to pain sensation but also to the inflammatory response.

The SNX25-NGF Axis in Pain Sensitivity

To really tease out how SNX25 and NGF were working, the team began examining levels of NGF in Snx25-deficient (Snx25+/−) and WT mice. NGF has a known role in regulating pain through its impact on certain genes such as Trpv1 and Scn9a. First off, the researchers noticed that the NGF levels were reduced in the skin of Snx25+/− mice compared to WT mice, both at rest and after formalin injection. NGF was also less abundant in dermatomal macrophages in Snx25+/− mice than in WT mice.

By injection of native mouse NGF 2.5S protein (99%) (#N-240) (Alomone Labs) into the right hind paw, the researches demonstrated restored pain sensitivity in Snx25+/− mice (figure 2).

Figure 2. VF thresholds before and 24 h after injection in WT or Snx25+/− mice injected with NGF (10 ng μl−1, 10 μl) or PBS (NGF, WT, n = 5, P = 0.017; Snx25+/−, n = 7, P = 0.014. PBS, WT, n = 4; Snx25+/−, n = 5). g, gram. Results are represented as mean ± s.e.m. Tanaka, T et al. Nat Immunol 24, 439–451 (2023).

Next, they focused on the molecular underpinnings linking SNX25 to Ngf mRNA production and found that knocking down Nrf2 led to a reduction in Ngf mRNA. Further investigation revealed that SNX25 appears to interfere with the ubiquitination of Nrf2, thereby influencing Ngf mRNA levels. Indeed, higher levels of poly-ubiquitinated Nrf2 were detected in the absence of SNX25. Taken together, this suggests a regulatory loop where SNX25 modulates Nrf2 ubiquitination to influence Ngf mRNA levels.

The Intricate Interplay Between Macrophages and Nerves

Macrophages do more than eat up cellular debris; they also play a pivotal role in nerve maintenance and pain sensitivity. This detailed investigation, which came off the back of serendipitous observation, provides new data regarding the essential role of the SNX25–Nrf2 axis in controlling pain sensitivity. Specifically, Snx25+/− mice show diminished pain responses under both normal and painful conditions. The underlying biochemistry involves SNX25-mediated protective action against Nrf2 ubiquitination and subsequent degradation, crucial for maintaining NGF production in dermatomal macrophages.

Potential Therapeutic Implications and Future Directions

Beyond academic merit, these results indicate a pathway that could bridge the gap between hyperalgesic conditions and conditions like HSAN V, where there’s a marked loss of pain sensitivity due to NGF gene mutations. Despite setbacks in developing NGF-neutralizing antibodies for clinical use, mainly due to adverse side effects, these findings highlight the SNX25–Nrf2 axis as a promising alternative for pain management. 

However, the complete picture of how peripheral NGF levels are regulated is still unclear. As NGF is also produced by non-inflammatory cells like keratinocytes, future studies must cast a wider net to capture the full range of cellular and molecular mechanisms in play. It’s going to be very interesting to see where this research goes next.

\Neurotrophins/Growth Factors