MESENCEPHALIC ASTROCYTE-DERIVED NEUROTROPHIC FACTOR (MANF) FOR PREVENTING AND TREATING PERIPHERAL NEUROPATHIES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63/394,770, filed August 3, 2022, the contents of which are hereby incorporated by reference.

BACKGROUND

[0002] The disclosures of all publications, patents, patent application publications and books referred to in this application are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.

[0003] The innate immune system plays an important role in driving neuropathic pain and neurodegeneration in chemotherapy-induced peripheral neuropathy (CIPN) (1-12). Tn particular, macrophages, white blood cells that are sentinels of tissue maintenance and involved in phagocytosis of damaged or foreign cells, are involved in all steps of pain, from onset to resolution (13-15). Macrophages can increase hypersensitivity in response to chemotherapeutics because systemic inhibition of pro-inflammatory macrophage activation and infiltration by minocycline or chemokine MCP-l/CCL-2 neutralizing antibodies protects sensory neurons from developing neuropathic pain and degeneration of peripheral nerves in the skin (1,4,11,12). However, macrophages not only control inflammation but promote subsequent regeneration of tissues by clearing cellular debris, and by promoting angiogenesis, and the extracellular matrix formation (16,17). Inflamed macrophages also contribute to paclitaxel (PTX)-mediated cancer treatment by infiltrating and killing tumors (18-20). As such, the responses of macrophages to chemotherapeutics can alter cellular processes and pain experiences in complex ways. Furthermore, molecular profiles of macrophages change continuously depending on their extracellular environment as they transition from resident to activated states: either pro- inflammatory and pain-producing or anti-inflammatory and analgesic forms (14,17,21-24). Many intermediate forms of activated macrophages exist in-between these two extremes (14,23,25-27), further confounding the potential use of macrophage-based therapies for patients suffering from CIPN. To safely target macrophages for CIPN treatment, it is imperative to better understand how different macrophage activation states contribute to the pathological progression of neuropathic pain. However, how these local macrophages respond to chemotherapeutics and contribute to CIPN is poorly understood.

[0004] The present invention addresses these needs and provides compositions and methods for treating peripheral neuropathies including treatment of taxane-induced peripheral neuropathies.

SUMMARY OF THE INVENTION

[0005] A method of preventing, reducing development of, or treating a peripheral neuropathy in a subject is provided, comprising administering to the subject an amount of a mesencephalic astrocyte-derived neurotrophic factor (MANF), or a nucleic acid encoding therefor, effective to treat a peripheral neuropathy.

[0006] Also provided is a method of reducing taxane-induced macrophage activation in a subject receiving a taxane treatment, or who is to receive a taxane treatment, comprising administering to the subject an amount of a mesencephalic astrocyte-derived neurotrophic factor (MANF), or nucleic acid encoding therefor, effective to treat a peripheral reducing taxane- induced macrophage activation in a subject.

[0007] Also provided is a method of reducing peripheral pain in a subject associated with activated pro-inflammatory macrophages comprising locally administering to the area of peripheral pain an amount of a mesencephalic astrocyte-derived neurotrophic factor (MANF), or nucleic acid encoding therefor, effective to reduce the peripheral pain.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Fig 1. Live imaging of PTX-treated animals reveals the timeline of morphological degeneration in nociceptive neurons in Drosophila. Yellow asterisks (varicosities and fragmentation, quantified as # degeneration in 243x243pm ² ). Error bars=SD. One-way ANOVA (w/Tukey’s posttest) VC=VehCont, P=PTX 20pM.

[0009] Fig 2. Macrophage-like cell transition in Drosophila models following cellular stress. PM=plasmatocyte, LM=lamellocyte, Green=Eater, Red= MSN, Magenta = integrin [IPS.

[0010] Fig. 3. Macrophage surrounding nociceptive neurons activates and transitions in response to PTX (20pM) insult in a time-dependent manner in larvae. 2nd row: enlarged insets.

[0011] Fig 4. A pilot single-cell Drosophila macrophage quantification in situ surrounding nociceptive neurons showed dose-dependent transition of hemocytes at 48h. Single cell segmentation by an in-house trained deep-learning algorithm. VehCont n=33, PTX 5pM n=182, PTX 20pM n=670 (n= number of hemocytes/ Drosophila macrophages in a nociceptive neuron receptive field tested).

[0012] Fig. 5A-5D. Macrophages are closely localized to nociceptive terminals in Drosophila. 5 A control. Green=nociceptive neurons, Eater; Red=MSN. 5B. Inset shows ruffling (arrowheads) and vacuoles (asterisks). 5C. Transition to LM. 5D. Arrowheads point to neurites wrapping a macrophage cluster.

[0013] Fig. 6. MANF overexpression protects nociceptive terminal morphology from PTX in Drosophila. Chronic treatment (20pM), arrow heads indicate degeneration. Two-way ANOVA (w/ Bonferroni posttest). Error bars = SEM

[0014] Fig. 7. MANF protects PTX-induced toxicity in adult DRG neurons in vitro. PTX 50nM 72h ± MANF (200nM). NF200. Tukey’s box and whiskers. Left: Kruskal -Wallis test (w/Dunn’s posttest), Right: 1-way ANOVA (w/Tukey’s posttest). n=6 ROIs (1.3x1.3mm ² ) each. Green asterisks= cell bodies.

[0015] Fig. 8. DRG neuron-macrophage co-culture. Compartment microfluidic chamber to study peripheral interaction between axon terminals (green) and macrophages (MP; red and blue). DETAILED DESCRIPTION

[0016] A method of preventing, reducing development of, or treating a peripheral neuropathy in a subject is provided, comprising administering to the subject an amount of a mesencephalic astrocyte-derived neurotrophic factor (MANF), or a nucleic acid encoding therefor, effective to treat a peripheral neuropathy.

[0017] In embodiments, the peripheral neuropathy is a chemotherapy-induced peripheral neuropathy (CIPN).

[0018] In embodiments, the peripheral neuropathy is a taxane-induced peripheral neuropathy.

[0019] In embodiments, the peripheral neuropathy is a paclitaxel-induced peripheral neuropathy.

[0020] In embodiments, the method is of preventing or reducing the reducing development of the CIPN and the MANF, or nucleic acid encoding therefor, is administered prior to the subject receiving chemotherapy.

[0021] In embodiments, the method further comprises administering MANF, or nucleic acid encoding therefor, to the subject during a period of chemotherapy administration.

[0022] In embodiments, the method is of treating an extant CIPN and the MANF, or nucleic acid encoding therefor, is administered during and/or subsequent to the subject receiving chemotherapy.

[0023] In embodiments, the CIPN is acute transient CIPN.

[0024] In embodiments, the CIPN is sub-acute long-lasting CIPN.

[0025] In embodiments, the peripheral neuropathy is an idiopathic peripheral neuropathy.

[0026] In embodiments, the idiopathic peripheral neuropathy is an aging-related idiopathic peripheral neuropathy.

[0027] In embodiments, the peripheral neuropathy is a sensory small fiber peripheral neuropathy.

[0028] In embodiments, the sensory small fiber peripheral neuropathy is a pre-diabetic neuropathy, a diabetic neuropathy, an HIV neuropathy, an amyloid neuropathy, or an inflammatory neuropathy.

[0029] In embodiments, the peripheral neuropathy is a diabetic peripheral neuropathy.

[0030] In embodiments, the subject has type 1 diabetes. [0031] In embodiments, the subject has type 2 diabetes mellitus.

[0032] In embodiments, the MANF, or nucleic acid encoding therefor, is administered locally to a site of the peripheral neuropathy.

[0033] In embodiments, the MANF, or nucleic acid encoding therefor, is locally administered to hands and/or feet of the subject. In embodiments, the MANF, or nucleic acid encoding therefor, is injected into the hands or feet. In embodiments, the MANF, or nucleic acid encoding therefor, is injected into the leg of the subject.

[0034] In embodiments, the subject is not being treated with a non-taxane chemotherapy.

[0035] In embodiments, the methods further comprise diagnosing the subject as suffering from a taxane-induced peripheral chemotherapy or as susceptible to a taxane-induced peripheral chemotherapy, prior to administering the MANF, or nucleic acid encoding therefor, to the subject.

[0036] In embodiments, the methods further comprise diagnosing the subject as suffering from an idiopathic or sensory small fiber peripheral neuropathy or as susceptible to an idiopathic or sensory small fiber peripheral neuropathy, prior to administering the MANF, or nucleic acid encoding therefor, to the subject.

[0037] Also provided is a method of reducing taxane-induced macrophage activation in a subject receiving a taxane treatment, or who is to receive a taxane treatment, comprising administering to the subject an amount of a mesencephalic astrocyte-derived neurotrophic factor (MANF), or nucleic acid encoding therefor, effective to treat a peripheral reducing taxane- induced macrophage activation in a subject.

[0038] Also provided is a method of reducing peripheral pain in a subject associated with activated pro-inflammatory macrophages comprising locally administering to the area of peripheral pain an amount of a mesencephalic astrocyte-derived neurotrophic factor (MANF), or nucleic acid encoding therefor, effective to reduce the peripheral pain.

[0039] In embodiments, the MANF disclosed herein is lyophilized and/or freeze dried and are reconstituted for use. Compositions or pharmaceutical compositions comprising the MANF disclosed herein can comprise stabilizers to prevent loss of activity or structural integrity of the protein due to the effects of denaturation, oxidation or aggregation over a period of time during storage and transportation prior to use. The compositions or pharmaceutical compositions can comprise one or more of any combination of salts, surfactants, pH and tonicity agents such as sugars can contribute to overcoming aggregation problems. Where a composition or pharmaceutical composition of the present invention is used as an injection or infusion, it is desirable to have a pH value in an approximately neutral pH range, it is also advantageous to minimize surfactant levels to avoid bubbles in the formulation which are detrimental for injection into subjects. In an embodiment, the composition or pharmaceutical composition is in liquid form and stably supports high concentrations of M ANH in solution and is suitable for inhalational or parenteral administration. In an embodiment, the composition or pharmaceutical composition is suitable for intravenous, intramuscular, intraperitoneal, intradermal, intraorgan, and/or subcutaneous injection. In an embodiment, the composition or pharmaceutical composition is in liquid form and has minimized risk of bubble formation and anaphylactoid side effects. In an embodiment, the composition or pharmaceutical composition is isotonic. In an embodiment, the composition or pharmaceutical composition has a pH or 6.8 to 7.4.

[0040] Examples of pharmaceutically acceptable carriers include, but are not limited to, phosphate buffered saline solution, sterile water (including water for injection USP), emulsions such as oil/water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline, for example 0.9% sodium chloride solution, USP. Compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000, the content of each of which is hereby incorporated in its entirety). In non-limiting examples, the can comprise one or more of dibasic sodium phosphate, potassium chloride, monobasic potassium phosphate, polysorbate 80 (e.g. 2-[2-[3,5-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethox y)ethoxy]ethyl (E)-octadec- 9-enoate), disodium edetate dehydrate, sucrose, monobasic sodium phosphate monohydrate, and dibasic sodium phosphate dihydrate.

[0041] In an embodiment the composition or pharmaceutical composition comprising the MANF described herein is substantially pure with regard to the MANF. A composition or pharmaceutical composition comprising the MANF described herein is "substantially pure" with regard to the MANF when at least 60% to 75% of a sample of the composition or pharmaceutical composition exhibits a single species of the MANF. A substantially pure composition or pharmaceutical composition comprising the MANF described herein can comprise, in the portion thereof which is the MANF, 60%, 70%, 80% or 90% of the MANF, more usually about 95%, and preferably over 99%. Purity or homogeneity may be tested by a number of means well known in the art, such as polyacrylamide gel electrophoresis or HPLC.

[0042] In an embodiment, the MANF is a recombinantly-produced MANF. In an embodiment, the MANF has the sequence of a human MANF. In an embodiment, the MANF comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0043] In an embodiment the nucleic acid encoding MANF is an mRNA. In an embodiment, the nucleic acid encoding MANF is administered via a lipid nanoparticle composition comprising the mRNA.

[0044] Administration can be local. Administration can be performed in a manner so as to not elicit systemic effects. Administration can be intramuscular or subcutaneous. Administration can be via infusion or injection. Administration can also be direct to an affected body part where peripheral neuropathy symptoms are occurring or expected to occur. Administration can be via local injection. Administration can also be auricular, buccal, conjunctival, cutaneous, subcutaneous, endocervical, endosinusial, endotracheal, enteral, epidural, via hemodialysis, interstitial, intrab dominal, intraamniotic, intra-arterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intraci sternal, intracorneal, intracoronary, intradermal, intradiscal, intraductal, intraepidermal, intraesophagus, intragastric, intravaginal, intragingival, intraileal, intraluminal, intralesional, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intraepicardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intraorgan, intratympanic, intrauterine, intravascular, intravenous, intraventricular, intravesical, intravitreal, laryngeal, nasal, nasogastric, ophthalmic, oral, oropharyngeal, parenteral, percutaneous, periarticular, peridural, rectal, inhalationally, retrobulbar, subarachnoid, subconjuctival, sublingual, submucosal, topically, transdermal, transmucosal, transplacental, transtracheal, ureteral, uretheral, and vaginal.

[0045] Symptoms of peripheral neuropathy, that can be ameliorated or prevented by the methods herein, include one or more of numbness and tingling in the feet or hands; burning, stabbing or shooting pain in affected areas; loss of balance and co-ordination; muscle weakness, including in the feet or hands [0046] In embodiments, the MANF is administered at a dose of 0.1 pg/kg to 1 pig/kg. In embodiments, the MANF is administered at a dose of 0.5 pg/kg to 5 pg/kg. In embodiments, the MANF is administered at a dose of 1.5 pg/kg to 2.5 pg/kg. In embodiments, the MANF is administered at a dose of 0.5 pg/kg to 500 pg/kg. In embodiments, the MANF is administered at a dose of 0.5mg/kg to 100 mg/kg. In embodiments, the MANF is administered at a dose of 101 mg/kg to 250 mg/kg. In embodiments, the MANF is administered at a dose of 251 mg/kg to 500 mg/kg. In embodiments, the MANF is administered at a dose of 501 mg/kg to 1000 mg/kg. In embodiments, the MANF is administered at a dose of 1001 mg/kg to 2000 mg/kg. In embodiments, the MANF is administered at a dose indicated herein once per day, twice per daily, daily, every other day, weekly, monthly or every three months. In embodiments, the MANF is administered at a dose of 25 to 100 mg twice daily, daily, every other day, weekly, monthly or every three months. In embodiments, the MANF is administered at a dose of 1.5 pg/kg to 2.5 pg/kg twice daily, daily, every other day, weekly, monthly or every three months. In embodiments, the MANF is administered at a dose of 100 to 250 mg twice daily, daily, every other day, weekly, monthly or every three months. In embodiments, the MANF is administered at a dose of 250 to 500 mg twice daily, daily, every other day, weekly, monthly or every three months. In embodiments, the MANF is administered at a dose of 500 to 1000 mg twice daily, daily, every other day, weekly, monthly or every three months. In embodiments, the MANF is administered at a dose of 1000 to 2000 mg twice daily, daily, every other day, weekly, monthly or every three months.

[0047] Another aspect of the invention provides a method for the prevention, treatment or amelioration of a peripheral neuropathy. According to this aspect, the MANF composition as disclosed hereinabove is administered to a subject in need of such a prevention, treatment or amelioration.

[0048] In a preferred embodiment, the method of peripheral neuropathy prevention, treatment or amelioration occurs in a human.

[0049] “And/or” as used herein, for example, with option A and/or option B, encompasses the separate embodiments of (i) option A, (ii) option B, and (iii) option A plus option B.

[0050] All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or otherwise clearly contradicted by context. [0051] Definitions: The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the methods of the invention and how to use them. Moreover, it will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of the other synonyms. The use of examples anywhere in the specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of the invention or any exemplified term. Likewise, the invention is not limited to its preferred embodiments.

[0052] The term “subject” as used in this application means a mammal. Mammals include canines, felines, rodents, bovine, equines, porcines, ovines, and primates including humans. Thus, the invention can be used in human medicine or also in veterinary medicine, e.g., to treat companion animals, farm animals, laboratory animals in zoological parks, and animals in the wild. The invention is particularly desirable for human medical applications. In a preferred embodiment the subject is a human.

[0053] The terms “treat”, “treatment” of a disease, and the like refer to slowing down, relieving, ameliorating or alleviating at least one of the symptoms of the peripheral neuropathy, or reversing the disease after its onset.

[0054] The terms “prevent”, “prevention”, and the like refer to acting prior to overt disease or disorder onset, to prevent the disease or disorder from developing or minimize the extent of the peripheral neuropathy or slow its course of development.

[0055] The term “in need thereof’ with regard to a subject would be a subject known or suspected of having or being at risk of developing peripheral neuropathy.

[0056] The terms “therapeutically effective amount” or "amount effective to" encompasses an amount sufficient to ameliorate or prevent a symptom or sign of the medical condition. An effective amount for a particular subject may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side effects. An effective amount can be the maximal dose or dosing protocol that avoids significant side effects or toxic effects.

[0057] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system, i.e., the degree of precision required for a particular purpose, such as a pharmaceutical formulation. For example, “about” can mean within 1 or more than 1 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0058] In embodiments of the methods herein, the subject is a human subject.

[0059] In embodiments of the methods herein, the MANF comprises the amino acid sequence as set forth in SEQ ID NO:1 hereinbelow.

[0060] In embodiments of the methods herein, the MANF is administered.

[0061] In embodiments of the methods herein, the nucleic acid encoding MANF is administered. In embodiments of the methods herein, the nucleic acid comprises an mRNA encoding the MANF. In embodiments of the methods herein, the mRNA is administered in the form of a lipid nanoparticle composition comprising the mRNA.

[0062] In an embodiment, the MANF has the sequence of a human MANF. In an embodiment, the MANF comprises the following sequence:

LRPGDCEVCISYLGRFYQDLI<DRDVTFSPATIENELII<FCRFARGI< ;ENRLCYYIGATDDA ATKIINEVSKPLAHHIPVEKICEKLKKKDSQICELKYDKQIDLSTVDLKKLRVKELKKIL D DWGETCKGCAEKSDYIRKINELMPKYAPKAASARTDL (SEQ ID NO: 1).

[0063] In an embodiment, the MANF has the sequence of a human MANF set forth in Uniprot ID P55145. In an embodiment, the MANF is non-glycosylated. EXPERIMENTAL EXAMPLES

[0064] How macrophages transition between pro- and anti-inflammatory status in a

Drosophila CIPN model: Macrophages are one of the major drivers of painful CIPN (2,3,5,12,41), yet how they transition throughout pathological progression is poorly understood. Single-cell RNAseq analyses of Drosophila immune cells (26,27) and our initial results highlight the diversity of activated macrophages after cellular stress. Moreover, there are at least 14 different clusters of Drosophila immune cells that become differentially activated following wasp attack and injury (27). The mechanisms of macrophage activation in the context of sensory neuron pathology in CIPN will be characterized. Targets, such as mesencephalic astrocyte- derived neurotrophic factor (MANF), are identified to locally modulate immune responses (42) in CIPN. In addition, because many genes expressed in Drosophila macrophages are conserved in the mammalian system (43), the study provides an invaluable in vivo model to understand the pathological progression and specific treatment targets for CIPN.

[0065] Paclitaxel (PTX)-induced macrophage activation precedes neuronal degeneration in Drosophila in vivo: A recent study by this laboratory established a timeline of pathological stages in PTX-induced peripheral neuropathy in mouse and Drosophila CIPN models and revealed that intracellular changes precede morphological changes in sensory neuron degeneration. Initial changes in both models include a reduction in motility of recycling endosomes (Rab4 ⁺ ) and lysosomes (40) We identified early (24h), intermediate (48h), and late (72h) pathological stages in our Drosophila CIPN model (Fig. 1). Behavioral studies in the literature (24 and 48h) (44) and our preliminary analysis at 72h support an emergence of hypersensitivity to noxious heat during pathological progression.

[0066] Administration of recombinant human MANF proteins protects DRG neurons from PTX toxicity: Due to MANF’s highly conserved role in Drosophila and mammalian models (42,56,59), we tested the protective capacity of MANF in an adult mouse CIPN model optimized in our previous study (40). We administered recombinant human (rh) MANF proteins in DRG culture and compared neuronal integrity and growth to vehicle controls lacking MANF supplementation. Initial results disclosed herein indicate that MANF protects from PTX-induced degeneration and neurite reduction (Fig. 7), supporting a conserved protective role for MANF. Because MANF is directly involved in immune cell differentiation and anti-inflammatory status (42,59,61,74), the effect of the MANF-mediated protection is also directly applicable to macrophages.

[0067] REFERENCES

[0068] 1 Huang, Z. Z. et al. CX3CL1 -mediated macrophage activation contributed to paclitaxel-induced DRG neuronal apoptosis and painful peripheral neuropathy. Brain Behav Immun e, 155-165, doi: 10.1016/j .bbi.2014.03.014 (2014).

[0069] 2 Kiguchi, N. et al. The critical role of invading peripheral macrophage- derived interleukin-6 in vincristine-induced mechanical allodynia in mice. Eur J Pharmacol 592, 87-92, doi:10.1016/j.ejphar.2008.07.008 (2008).

[0070] 3 Liu, C. et al. Upregulation of CCL2 via ATF3/c-Jun interaction mediated the

Bortezomib -induced peripheral neuropathy. Brain Behav Immun 53, 96-104, doi:10.1016/j.bbi.2015.11.004 (2016).

[0071] 4 Liu, C. C. et al. Prevention of paclitaxel-induced allodynia by minocycline: Effect on loss of peripheral nerve fibers and infiltration of macrophages in rats. Mol Pain 6, 76, doi : 10.1186/1744-8069-6-76 (2010).

[0072] 5 Makker, P. G. et al. Characterisation of Immune and Neuroinflammatory Changes

Associated with Chemotherapy-Induced Peripheral Neuropathy. PLoS One 12, e0170814, doi:10.1371/journal. pone.0170814 (2017).

[0073] 6 Meregalli, C. et al. High-dose intravenous immunoglobulins reduce nerve macrophage infiltration and the severity of bortezomib-induced peripheral neurotoxicity in rats. J Neuroinflammation 15, 232, doi: 10.1186/sl2974-018-1270-x (2018).

[0074] 7 Montague, K. & Malcangio, M. The Therapeutic Potential of

Monocyte/Macrophage Manipulation in the Treatment of Chemotherapy-Induced Painful Neuropathy. Frontiers in Molecular Neuroscience 10 (2017).

[0075] 8 Peters, C. M. et al. Intravenous paclitaxel administration in the rat induces a peripheral sensory neuropathy characterized by macrophage infiltration and injury to sensory neurons and their supporting cells. Exp Neurol 203, 42-54, doi:10.1016/j.expneurol.2006.07.022 (2007).

[0076] 9 Shepherd, A. J. et al. Angiotensin II Triggers Peripheral Macrophage-to-Sensory Neuron Redox Crosstalk to Elicit Pain. J Neurosci 38, 7032-7057, doi:10.1523/JNEUROSCI.3542-17.2018 (2018).

[0077] 10 Starobova, H. et al. Vincristine-induced peripheral neuropathy is driven by canonical NLRP3 activation and IL- 1(1 release. The Journal of experimental medicine 218 5 (2021).

[0078] 11 Starobova, H. et al. Minocycline Prevents the Development of Mechanical

Allodynia in Mouse Models of Vincristine-Induced Peripheral Neuropathy. Frontiers in Neuroscience 13 (2019). [0079] 12 Zhang, H. et al. Dorsal Root Ganglion Infiltration by Macrophages Contributes to

Paclitaxel Chemotherapy-Induced Peripheral Neuropathy. J Pain 17, 775-786, doi:10.1016/j.jpain.2016.02.011 (2016).

[0080] 13 Celik, M. O., Labuz, D., Keye, J., Glauben, R. & Machelska, H. LL-4 induces M2 macrophages to produce sustained analgesia via opioids. JCI Insight 5, doi: 10.1172/jci.insight.133093 (2020).

[0081] 14 Chen, O , Donnelly, C. R. & Ji, R. R. Regulation of pain by neuro-immune interactions between macrophages and nociceptor sensory neurons. Curr Opin Neurobiol 62, 17- 25, doi:10.1016/j.conb.2019.11.006 (2020).

[0082] 15 Pannell, M. et al. Adoptive transfer of M2 macrophages reduces neuropathic pain via opioid peptides. J Neuroinflammation 13, 262, doi: 10.1186/sl2974-016-0735-z (2016).

[0083] 16 Oishi, Y. & Manabe, I. Macrophages in inflammation, repair and regeneration. Int

Immunol 30, 511-528, doi: 10.1093/intimm/dxy054 (2018).

[0084] 17 Mahdavian Delavary, B., van der Veer, W. M., van Egmond, M., Niessen, F. B. &

Beelen, R. H. Macrophages in skin injury and repair. Immunobiology 216, 753-762, doi:10.1016/j.imbio.2011.01.001 (2011).

[0085] 18 Shen, J., Chen, C., Li, Z. & Hu, S. Paclitaxel Promotes Tumor-Infiltrating

Macrophages in Breast Cancer. Technol Cancer Res Treat 19, 1533033820945821, doi:10.1177/1533033820945821 (2020).

[0086] 19 Wanderley, C. W. et al. Paclitaxel Reduces Tumor Growth by Reprogramming

Tumor-Associated Macrophages to an Ml Profile in a TLR4-Dependent Manner. Cancer Res 78, 5891-5900, doi: 10.1158/0008-5472. CAN-17-3480 (2018).

[0087] 20 Yamaguchi, T. et al. Low-dose paclitaxel suppresses the induction of M2 macrophages in gastric cancer. Oncol Rep 37 , 3341-3350, doi:10.3892/or.2017.5586 (2017).

[0088] 21 Brazil, J. C., Quiros, M., Nusrat, A. & Parkos, C. A. Innate immune cell-epithelial crosstalk during wound repair. J Clin Invest 129, 2983-2993, doi: 10.1172/JCI124618 (2019).

[0089] 22 DiPietro, L. A., Wilgus, T. A. & Koh, T. J. Macrophages in Healing Wounds:

Paradoxes and Paradigms. Ini J Mol Sci 22, doi:10.3390/ijms22020950 (2021).

[0090] 23 Kolter, J. et al. A Subset of Skin Macrophages Contributes to the Surveillance and

Regeneration of Local Nerves. Immunity 50, 1482-1497 el487, doi:10.1016/j.immuni.2019.05.009 (2019).

[0091] 24 Bang, S. et al. GPR37 regulates macrophage phagocytosis and resolution of inflammatory pain. J Clin Invest 128, 3568-3582, doi: 10.1172/JCI99888 (2018).

[0092] 25 Anderl, I. et al. Transdifferentiation and Proliferation in Two Distinct Hemocyte

Lineages in Drosophila melanogaster Larvae after Wasp Infection. PLoS Pathog 12, el005746, doi : 10.1371 /j ournal .ppat.1005746 (2016) . [0093] 26 Cho, B. et al. Single-cell transcriptome maps of myeloid blood cell lineages in

Drosophila. Nat Commun 11, 4483, doi: 10.1038/s41467-020-18135-y (2020).

[0094] 27 Tattikota, S. G. et al. A single-cell survey of Drosophila blood. Elife 9, doi:10.7554/eLife.54818 (2020).

[0095] 28 Hall, D. H. & Treinin, M. How does morphology relate to function in sensory arbors? Trends Neurosci 34, 443-451, doi: 10.1016/j. tins.2011.07.004 (2011).

[0096] 29 Hwang, R. Y. et al. Nociceptive neurons protect Drosophila larvae from parasitoid wasps. Curr Biol 17, 2105-2116, doi: 10.1016/j. cub.2007.11.029 (2007).

[0097] 30 Poe, A. R. et al. Dendritic space-filling requires a neuronal type-specific extracellular permissive signal in Drosophila. P Natl Acad Sci USA 114, E8062-E8071, doi : 10.1073/pnas.1707467114 (2017).

[0098] 31 Wendel schafer-Crabb, G., Kennedy, W. R. & Walk, D. Morphological features of nerves in skin biopsies. J Neurol Sci 242, 15-21, doi: 10.1016/j.jns.2005.11.010 (2006).

[0099] 32 Kim, M. E., Shrestha, B. R., Blazeski, R., Mason, C. A. & Grueber, W. B.

Integrins establish dendrite-substrate relationships that promote dendritic self-avoidance and patterning in drosophila sensory neurons. Neuron 73, 79-91, doi: 10.1016/j. neuron.2011.10.033 (2012).

[00100] 33 Han, C. et al. Integrins regulate repulsion-mediated dendritic patterning of drosophila sensory neurons by restricting dendrites in a 2D space. Neuron 73, 64-78, doi: 10.1016/j. neuron.201 1.10.036 (2012).

[00101] 34 Shin, M., Cha, N., Koranteng, F., Cho, B. & Shim, J. Subpopulation of

Macrophage-Like Plasmatocytes Attenuates Systemic Growth via JAK/STAT in the Drosophila Fat Body. Front Immunol 11, 63, doi: 10.3389/fimmu.2020.00063 (2020).

[00102] 35 Weavers, H., Evans, I. R , Martin, P. & Wood, W. Corpse Engulfment Generates a Molecular Memory that Primes the Macrophage Inflammatory Response. Cell 165, 1658-1671, doi:10.1016/j. cell.2016.04.049 (2016).

[00103] 36 Makhijani, K. et al. Regulation of Drosophila hematopoietic sites by Activin-beta from active sensory neurons. Nat Commun 8, 15990, doi: 10.1038/ncommsl5990 (2017).

[00104] 37 Makhijani, K., Alexander, B., Tanaka, T., Rulifson, E. & Bruckner, K. The peripheral nervous system supports blood cell homing and survival in the Drosophila larva. Development 138, 5379-5391, doi: 10.1242/dev.067322 (2011).

[00105] 38 Stahl, P. L. et al. Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science 353, 78-82, doi: 10.1126/science.aaf2403 (2016).

[00106] 39 Akin, E. J. et al. Paclitaxel increases axonal localization and vesicular trafficking of Navl.7. 5raz>z 144, 1727-1737, doi:10.1093/brain/awabl 13 (2021). [00107] 40 Shin, G. J. et al. Tntegrins protect sensory neurons in models of paclitaxel-induced peripheral sensory neuropathy. Proc Natl Acad Set U S A 118, doi: 10.1073/pnas.2006050118 (2021).

[00108] 41 Vichaya, E. G. et al. Mechanisms of chemotherapy-induced behavioral toxicides.

Frontiers in Neuroscience 9 (2015).

[00109] 42 Neves, J. et al. Immune modulation by MANF promotes tissue repair and regenerative success in the retina. Science 353, aaf3646, doi : 10.1126/science.aaf3646 (2016).

[00110] 43 Baneijee, U., Girard, J. R., Goins, L. M. & Spratford, C. M. Drosophila as a

Genetic Model for Hematopoiesis. Genetics 211, 367-417, doi:10.1534/genetics.118.300223 (2019).

[00111] 44 Brazill, J. M., Cruz, B., Zhu, Y. & Zhai, R. G. Nmnat mitigates sensory dysfunction in a Drosophila model of paclitaxel-induced peripheral neuropathy. Dis Model Meeh 11, doi: 10.1242/dmm.032938 (2018).

[00112] 45 Gold, K. S. & Bruckner, K. Macrophages and cellular immunity in Drosophila melanogaster. Semin Immunol 27, 357-368, doi:10.1016/j.smim.2016.03.010 (2015).

[00113] 46 Tokusumi, Y., Tokusumi, T. & Schulz, R. A. The nociception genes painless and

Piezo are required for the cellular immune response of Drosophila larvae to wasp parasitization. Biochem Biophys Res Commun 486, 893-897, doi: 10.1016/j.bbrc.2017.03.116 (2017).

[00114] 47 York, A. G. et al. Instant super-resolution imaging in live cells and embryos via analog image processing. Nat Methods 10, 1122-1 126, doi : 10.1038/nmeth.2687 (2013).

[00115] 48 Elkington, P. T., O'Kane, C. M. & Friedland, J. S. The paradox of matrix metalloproteinases in infectious disease. Clin Exp Immunol 142, 12-20, doi: 10.1111/j .1365- 2249.2005.02840.x (2005).

[00116] 49 Kano, S. I. et al. Glutathione S-transferases promote proinflammatory astrocytemicroglia communication during brain inflammation. Sci Signal 12, doi : 10.1126/sci signal . aar2124 (2019) .

[00117] 50 Ufer, F. et al. NvdAr SA governs inflammatory dendritic cell migration from the skin and thereby controls T cell activation. Sci Immunol 1, eaaf8665, doi: 10.1126/sciimmunol.aaf8665 (2016).

[00118] 51 Dorrington, M. G. & Fraser, I. D. C. NF-kappaB Signaling in Macrophages: Dynamics, Crosstalk, and Signal Integration. Front Immunol 10, 705, doi: 10.3389/fnnmu.2019.00705 (2019).

[00119] 52 Bernstein, K. E. et al. Angiotensin-converting enzyme in innate and adaptive immunity. Nat Rev Nephrol 14, 325-336, doi: 10.1038/nmeph.2018.15 (2018).

[00120] 53 Holbrook, J., Lara-Reyna, S., Jarosz-Griffiths, H. & McDermott, M. Tumour necrosis factor signalling in health and disease. FlOOORes 8, doi : 10.12688/f 1 OOOresearch.17023.1 (2019). [00121] 54 Petraki, S., Alexander, B. & Bruckner, K. Assaying Blood Cell Populations of the

Drosophila melanogaster Larva. J Vis Exp, doi: 10.3791/52733 (2015).

[00122] 55 Luo, X. et al. Macrophage Toll -like Receptor 9 Contributes to Chemotherapy - Induced Neuropathic Pain in Male Mice. J Neurosci 39, 6848-6864, doi:10.1523/JNEUROSCI.3257-18.2019 (2019).

[00123] 56 Lindahl, M., Saarma, M. & Lindholm, P. Unconventional neurotrophic factors

CDNF and MANF: Structure, physiological functions and therapeutic potential. Neurobiol Dis 97, 90-102, doi: 10.1016/j.nbd.2016.07.009 (2017).

[00124] 57 Matlik, K. et al. Role of two sequence motifs of mesencephalic astrocyte-derived neurotrophic factor in its survival-promoting activity. Cell Death Dis 6, e2032, doi : 10.1038/cddis.2015.371 (2015).

[00125] 58 Sousa- Victor, P., Jasper, H. & Neves, J. F. Trophic Factors in Inflammation and

Regeneration: The Role of MANF and CDNF. Frontiers in Physiology 9 (2018).

[00126] 59 Sousa- Victor, P. et al. MANF regulates metabolic and immune homeostasis in ageing and protects against liver damage. Nat Metab 1, 276-290, doi:10.1038/s42255-018-0023- 6 (2019).

[00127] 60 Tseng, K. Y. et al. MANF Is Essential for Neurite Extension and Neuronal

Migration in the Developing Cortex. eNeuro 4, doi:10.1523/ENEURO.0214-17.2017 (2017).

[00128] 61 Zhao, H. et al. Mesencephalic astrocyte-derived neurotrophic factor inhibits oxygen-glucose deprivation-induced cell damage and inflammation by suppressing endoplasmic reticulum stress in rat primary astrocytes. J Mol Neurosci 51, 671-678, doi: 10.1007/sl2031-013- 0042-4 (2013).

[00129] 62 Eesmaa, A. et al. The cytoprotective protein MANF promotes neuronal survival independently from its role as a GRP78 cofactor. J Biol Chem 296, 100295, doi:10.1016/j.jbc.202L 100295 (2021).

[00130] 63 Lindholm, P. et al. MANF is widely expressed in mammalian tissues and differently regulated after ischemic and epileptic insults in rodent brain. Mol Cell Neurosci 39, 356-371, doi:10.1016/j.mcn.2008.07.016 (2008).

[00131] 64 Petrova, P. et al. MANF: a new mesencephalic, astrocyte-derived neurotrophic factor with selectivity for dopaminergic neurons. J Mol Neurosci 20, 173-188, doi: 10.1385/jmn:20:2: 173 (2003).

[00132] 65 Gemer, M. Y., Kastenmuller, W ., Ifrim, I., Kabat, J. & Germain, R. N. Histo- cytometry: a method for highly multiplex quantitative tissue imaging analysis applied to dendritic cell subset microanatomy in lymph nodes. Immunity’ 37, 364-376, doi:10.1016/j.immuni.2012.07.011 (2012).

[00133] 66 Starkweather, A. Increased interleukin-6 activity associated with painful chemotherapy -induced peripheral neuropathy in women after breast cancer treatment. Nurs Res Pract 2010, 281531, doi : 10.1155/2010/281531 (2010). [00134] 67 Wang, X. M., Lehky, T. J., Brell, J. M. & Dorsey, S. G. Discovering cytokines as targets for chemotherapy-induced painful peripheral neuropathy. Cytokine 59, 3-9, doi:10.1016/j.cyto.2012.03.027 (2012).

[00135] 68 Zhou, Y. Q. et al. Interleukin-6: an emerging regulator of pathological pain. J

Neuroinflammation 13, 141, doi: 10.1186/sl2974-016-0607-6 (2016).

[00136] 69 Walkowicz, L. et al. Downregulation of DmMANF in Glial Cells Results in

Neurodegeneration and Affects Sleep and Lifespan in Drosophila melanogaster. Front Neurosci 11, 610, doi: 10.3389/fnins.2017.00610 (2017).

[00137] 70 Li, H. et al. Fly Cell Atlas: a single-cell transcriptomic atlas of the adult fruit fly. bioRxiv, doi: 10.1101/2021.07.04.451050 (2021). Accessed on 10/4/21.

[00138] 71 Airavaara, M. et al. Mesencephalic astrocyte-derived neurotrophic factor reduces ischemic brain injury and promotes behavioral recovery in rats. J Comp Neurol 515, 116-124, doi: 10.1002/cne.22039 (2009).

[00139] 72 Glembotski, C. C. et al. Mesencephalic astrocyte-derived neurotrophic factor protects the heart from ischemic damage and is selectively secreted upon sarco/endoplasmic reticulum calcium depletion. J Biol Chem 287, 25893-25904, doi: 10.1074/jbc.Ml 12.356345 (2012).

[00140] 73 Yang, W. et al. Mesencephalic astrocyte-derived neurotrophic factor prevents neuron loss via inhibiting ischemia-induced apoptosis. J Neurol Sci 344, 129-138, doi:10.1016/j.jns.2014.06.042 (2014).

[00141] 74 Hou, C. et al. MANF regulates splenic macrophage differentiation in mice.

Immunol Lett 212, 37-45, doi: 10.1016/j.imlet.2019.06.007 (2019).

[00142] 75 Hockley, J. R. et al. Multiple roles for NaV1.9 in the activation of visceral afferents by noxious inflammatory, mechanical, and human disease-derived stimuli. Pain 155, 1962-1975, doi: 10 1016/j.pain.2014.06.015 (2014).

[00143] 76 Khan, I. Z. et al. The CD200-CD200R Axis Promotes Squamous Cell Carcinoma

Metastasis via Regulation of Cathepsin K. Cancer Res 81, 5021-5032, doi: 10.1158/0008- 5472.CAN-20-3251 (2021).

[00144] 77 Meng, J. et al. Duloxetine, a Balanced Serotonin-Norepinephrine Reuptake

Inhibitor, Improves Painful Chemotherapy-Induced Peripheral Neuropathy by Inhibiting Activation of p38 MAPK and NF-kappaB. Front Pharmacol 10, 365, doi: 10.3389/fphar.2019.00365 (2019).

[00145] 78 Li, Y. et al. MAPK signaling downstream to TLR4 contributes to paclitaxel- induced peripheral neuropathy. Brain Behav Immun 49, 255-266, doi : 10.1016/j .bbi.2015.06.003 (2015).

[00146] 79 Wang, J. et al. Upregulation of CX3CL1 mediated by NF-kappaB activation in dorsal root ganglion contributes to peripheral sensitization and chronic pain induced by oxaliplatin administration. Mol Pain 13, 1744806917726256, doi: 10.1177/1744806917726256 (2017).

[00147] 80 Zhong, S. et al. Ketogenic diet prevents paclitaxel-induced neuropathic nociception through activation of PPARgamma signalling pathway and inhibition of neuroinflammation in rat dorsal root ganglion. Eur J Neurosci 54, 5341-5356, doi: 10.1111/ejn.15397 (2021).

[00148] 81 Sekiguchi, F. et al. Paclitaxel-induced HMGB1 release from macrophages and its implication for peripheral neuropathy in mice: Evidence for a neuroimmune crosstalk. Neuropharmacology 141, 201-213, doi: 10.1016/j. neuropharm.2018.08.040 (2018).

[00149] 82 Liu, J. et al. Mesencephalic Astrocyte-Derived Neurotrophic Factor Inhibits Liver

Cancer Through Small Ubiquitin-Related Modifier (SUMO)ylation-Related Suppression of NF- kappaB/Snail Signaling Pathway and Epithelial-Mesenchymal Transition. Hepatology 71, 1262- 1278, doi: 10.1002/hep.30917 (2020).

[00150] 83 Yagi, T. et al. Neuroplastin Modulates Anti-inflammatory Effects of MANF. iScience 23, 101810, doi: 10.1016/j isci.2020.101810 (2020).

[00151] 84 Wen, W. et al. Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF)

Regulates Neurite Outgrowth Through the Activation of Akt/mTOR and Erk/mTOR Signaling Pathways. Front Mol Neurosci 13, 560020, doi: 10.3389/fnmol.2020.560020 (2020).

[00152] 85 Zhang, J. et al. Nrf2-mediated neuroprotection by MANF against 6-0HDA- induced cell damage via PI3K/AKT/GSK3beta pathway. Exp Gerontol 100, 77-86, doi:10.1016/j.exger.2017.10.021 (2017).

[00153] 86 Figlia, G , Norrmen, C., Pereira, J. A., Gerber, D. & Suter, U. Dual function of the

PI3K-Akt-mTORCl axis in myelination of the peripheral nervous system. Elife 6, doi : 10.7554/eLife.29241 (2017).

[00154] 87 Giacoppo, S., Pollastro, F., Grassi, G , Bramanti, P. & Mazzon, E. Target regulation of PI3K/ Akt/mTOR pathway by cannabidiol in treatment of experimental multiple sclerosis. Fitoterapia 116, 77-84, doi: 10.1016/j .fitote.2016.11.010 (2017).

[00155] 88 Cho, C., Michailidis, V. & Martin, L. J. Revealing brain mechanisms of mTOR- mediated translational regulation: Implications for chronic pain. Neurobiol Pain 4, 27-34, doi : 10.1016/j .ynpai .2018.03.002 (2018).

[00156] 89 Chen, S. P. et al. PI3K/Akt Pathway: A Potential Therapeutic Target for Chronic

Pain. Curr Pharm Des 23, 1860-1868, doi: 10.2174/1381612823666170210150147 (2017).

[00157] 90 Vergadi, E., leronymaki, E., Lyroni, K., Vaporidi, K. & Tsatsanis, C. Akt

Signaling Pathway in Macrophage Activation and M1/M2 Polarization. J Immunol 198, 1006- 1014, doi:10.4049/jimmunol,1601515 (2017).

[00158] 91 Lisse, T. S. et al. Paclitaxel-induced epithelial damage and ectopic MMP-13 expression promotes neurotoxicity in zebrafish. Proc Natl Acad Sci U S A 113, E2189-2198, doi: 10.1073/pnas.1525096113 (2016). [00159] 92 Sadler, K. E. et al. Transient receptor potential canonical 5 mediates inflammatory mechanical and spontaneous pain in mice. Sci Transl Med 13, doi: 10.1126/scitranslmed.abd7702 (2021).

[00160] 93 Kerckhove, N. et al. Long-Term Effects, Pathophysiological Mechanisms, and

Risk Factors of Chemotherapy-Induced Peripheral Neuropathies: A Comprehensive Literature Review. Frontiers in Pharmacology 8, doi: 10.3389/fphar.2017.00086 (2017).

[00161] 94 Shah, A. et al. Incidence and disease burden of chemotherapy-induced peripheral neuropathy in a population-based cohort. J Neurol Neurosurg Psychiatry 89, 636-641, doi : 10.1136/j nnp-2017-317215 (2018).

[00162] 95 Catuneanu, A., Paylor, J. W., Winship, I., Colbourne, F. & Kerr, B. J. Sex differences in central nervous system plasticity and pain in experimental autoimmune encephalomyelitis. Pain 160, 1037-1049, doi: 10.1097/j. pain.0000000000001483 (2019).

[00163] 96 North, R. Y. et al. Electrophysiological and transcriptomic correlates of neuropathic pain in human dorsal root ganglion neurons. Brain 142, 1215-1226, doi:10.1093/brain/awz063 (2019).

[00164] 97 Tawfik, V. L. et al. Systematic Immunophenotyping Reveals Sex-Specific Responses After Painful Injury in Mice. Front Immunol 11, 1652, doi: 10.3389/fimmu.2020.01652 (2020).

[00165] 98 Sorge, R. E. et al. Spinal cord Toll-like receptor 4 mediates inflammatory and neuropathic hypersensitivity in male but not female mice. J Neurosci 31, 15450-15454, doi:10.1523/JNEUROSCI.3859-l 1.2011 (2011).

[00166] 99 Yu, X. et al. Dorsal root ganglion macrophages contribute to both the initiation and persistence of neuropathic pain. Nat Commun 11, 264, doi:10.1038/s41467-019-13839-2 (2020).

[00167] 100 Yang, S. et al. MANF regulates hypothalamic control of food intake and body weight. Nat Commun 8, 579, doi:10.1038/s41467-017-00750-x (2017).