Microglia Require CD4 T Cells to Complete the Fetal-to-Adult Transition.

The brain is a site of relative immune privilege. Although CD4 T cells have been reported in the central nervous system, their presence in the healthy brain remains controversial, and their function remains largely unknown. We used a combination of imaging, single cell, and surgical approaches to identify a CD69+ CD4 T cell population in both the mouse and human brain, distinct from circulating CD4 T cells. The brain-resident population was derived through in situ differentiation from activated circulatory cells and was shaped by self-antigen and the peripheral microbiome. Single-cell sequencing revealed that in the absence of murine CD4 T cells, resident microglia remained suspended between the fetal and adult states. This maturation defect resulted in excess immature neuronal synapses and behavioral abnormalities. These results illuminate a role for CD4 T cells in brain development and a potential interconnected dynamic between the evolution of the immunological and neurological systems. VIDEO ABSTRACT.

Astrocyte-targeted gene delivery of interleukin 2 specifically increases brain-resident regulatory T cell numbers and protects against pathological neuroinflammation.

The ability of immune-modulating biologics to prevent and reverse pathology has transformed recent clinical practice. Full utility in the neuroinflammation space, however, requires identification of both effective targets for local immune modulation and a delivery system capable of crossing the blood-brain barrier. The recent identification and characterization of a small population of regulatory T (Treg) cells resident in the brain presents one such potential therapeutic target. Here, we identified brain interleukin 2 (IL-2) levels as a limiting factor for brain-resident Treg cells. We developed a gene-delivery approach for astrocytes, with a small-molecule on-switch to allow temporal control, and enhanced production in reactive astrocytes to spatially direct delivery to inflammatory sites. Mice with brain-specific IL-2 delivery were protected in traumatic brain injury, stroke and multiple sclerosis models, without impacting the peripheral immune system. These results validate brain-specific IL-2 gene delivery as effective protection against neuroinflammation, and provide a versatile platform for delivery of diverse biologics to neuroinflammatory patients.

From dentistry to immunology: navigating challenges and building a career in neuroimmunology.

This Commentary recounts an academic journey from dentistry to neuroimmunology, highlighting pivotal moments such as a PhD fraught with challenges and an unexpected postdoctoral experience in France. My decision to settle in Belgium for a postdoc and subsequent transition to an assistant professorship at KU Leuven reflects resilience, adaptability and a commitment to both scientific exploration and family life. Balancing career uncertainties, motherhood and academic achievements, it encapsulates a trajectory shaped by a passion for neuroimmunology.

A Pilot Study to Develop Paraneoplastic Cerebellar Degeneration Mouse Model.

Modeling paraneoplastic neurological diseases to understand the immune mechanisms leading to neuronal death is a major challenge given the rarity and terminal access of patients' autopsies. Here, we present a pilot study aiming at modeling paraneoplastic cerebellar degeneration with Yo autoantibodies (Yo-PCD). Female mice were implanted with an ovarian carcinoma cell line expressing CDR2 and CDR2L, the known antigens recognized by anti-Yo antibodies. To boost the immune response, we also immunized the mice by injecting antigens with diverse adjuvants and immune checkpoint inhibitors. Ataxia and gait instability were assessed in treated mice as well as autoantibody levels, Purkinje cell density, and immune infiltration in the cerebellum. We observed the production of anti-Yo antibodies in the CSF and serum of all immunized mice. Brain immunoreaction varied depending on the site of implantation of the tumor, with subcutaneous administration leading to a massive infiltration of immune cells in the meningeal spaces, choroid plexus, and cerebellar parenchyma. However, we did not observe massive Purkinje cell death nor any motor impairments in any of the experimental groups. Self-sustained neuro-inflammation might require a longer time to build up in our model. Unusual tumor antigen presentation and/or intrinsic, species-specific factors required for pro-inflammatory engagement in the brain may also constitute strong limitations to achieve massive recruitment of antigen-specific T-cells and killing of antigen-expressing neurons in this mouse model.

CTLA4-Ig Effectively Controls Clinical Deterioration and Immune Condition in a Murine Model of Foxp3 Deficiency.

PURPOSE: FOXP3 deficiency results in severe multisystem autoimmunity in both mice and humans, driven by the absence of functional regulatory T cells. Patients typically present with early and severe autoimmune polyendocrinopathy, dermatitis, and severe inflammation of the gut, leading to villous atrophy and ultimately malabsorption, wasting, and failure to thrive. In the absence of successful treatment, FOXP3-deficient patients usually die within the first 2 years of life. Hematopoietic stem cell transplantation provides a curative option but first requires adequate control over the inflammatory condition. Due to the rarity of the condition, no clinical trials have been conducted, with widely unstandardized therapeutic approaches. We sought to compare the efficacy of lead therapeutic candidates rapamycin, anti-CD4 antibody, and CTLA4-Ig in controlling the physiological and immunological manifestations of Foxp3 deficiency in mice. METHOD: We generated Foxp3-deficient mice and an appropriate clinical scoring system to enable direct comparison of lead therapeutic candidates rapamycin, nondepleting anti-CD4 antibody, and CTLA4-Ig. RESULTS: We found distinct immunosuppressive profiles induced by each treatment, leading to unique protective combinations over distinct clinical manifestations. CTLA4-Ig provided superior breadth of protective outcomes, including highly efficient protection during the transplantation process. CONCLUSION: These results highlight the mechanistic diversity of pathogenic pathways initiated by regulatory T cell loss and suggest CTLA4-Ig as a potentially superior therapeutic option for FOXP3-deficient patients.

Regulatory T-cell stability and functional plasticity in health and disease.

FOXP3-expressing regulatory T cells (Treg ) are indispensable for immune homeostasis and tolerance, and in addition tissue-resident Treg have been found to perform noncanonical, tissue-specific functions. For optimal tolerogenic function during inflammatory disease, Treg are equipped with mechanisms that assure lineage stability. Treg lineage stability is closely linked to the installation and maintenance of a lineage-specific epigenetic landscape, specifically a Treg -specific DNA demethylation pattern. At the same time, for local and directed immune regulation Treg must possess a level of functional plasticity that requires them to partially acquire T helper cell (TH ) transcriptional programs-then referred to as TH -like Treg . Unleashing TH programs in Treg , however, is not without risk and may threaten the epigenetic stability of Treg with consequently pathogenic ex-Treg contributing to (auto-) inflammatory conditions. Here, we review how the Treg -stabilizing epigenetic landscape is installed and maintained, and further discuss the development, necessity and lineage instability risks of TH 1-, TH 2-, TH 17-like Treg and follicular Treg .

CD8 T cell-mediated killing of orexinergic neurons induces a narcolepsy-like phenotype in mice.

Narcolepsy with cataplexy is a rare and severe sleep disorder caused by the destruction of orexinergic neurons in the lateral hypothalamus. The genetic and environmental factors associated with narcolepsy, together with serologic data, collectively point to an autoimmune origin. The current animal models of narcolepsy, based on either disruption of the orexinergic neurotransmission or neurons, do not allow study of the potential autoimmune etiology. Here, we sought to generate a mouse model that allows deciphering of the immune mechanisms leading to orexin(+) neuron loss and narcolepsy development. We generated mice expressing the hemagglutinin (HA) as a "neo-self-antigen" specifically in hypothalamic orexin(+) neurons (called Orex-HA), which were transferred with effector neo-self-antigen-specific T cells to assess whether an autoimmune process could be at play in narcolepsy. Given the tight association of narcolepsy with the human leukocyte antigen (HLA) HLA-DQB1*06:02 allele, we first tested the pathogenic contribution of CD4 Th1 cells. Although these T cells readily infiltrated the hypothalamus and triggered local inflammation, they did not elicit the loss of orexin(+) neurons or clinical manifestations of narcolepsy. In contrast, the transfer of cytotoxic CD8 T cells (CTLs) led to both T-cell infiltration and specific destruction of orexin(+) neurons. This phenotype was further aggravated upon repeated injections of CTLs. In situ, CTLs interacted directly with MHC class I-expressing orexin(+) neurons, resulting in cytolytic granule polarization toward neurons. Finally, drastic neuronal loss caused manifestations mimicking human narcolepsy, such as cataplexy and sleep attacks. This work demonstrates the potential role of CTLs as final effectors of the immunopathological process in narcolepsy.

CTLA4 blockade elicits paraneoplastic neurological disease in a mouse model.

CTLA4 is an inhibitory regulator of immune responses. Therapeutic CTLA4 blockade enhances T cell responses against cancer and provides striking clinical results against advanced melanoma. However, this therapy is associated with immune-related adverse events. Paraneoplastic neurologic disorders are immune-mediated neurological diseases that develop in the setting of malignancy. The target onconeural antigens are expressed physiologically by neurons, and aberrantly by certain tumour cells. These tumour-associated antigens can be presented to T cells, generating an antigen-specific immune response that leads to autoimmunity within the nervous system. To investigate the risk to develop paraneoplastic neurologic disorder after CTLA4 blockade, we generated a mouse model of paraneoplastic neurologic disorder that expresses a neo -self antigen both in Purkinje neurons and in implanted breast tumour cells. Immune checkpoint therapy with anti-CTLA4 monoclonal antibody in this mouse model elicited antigen-specific T cell migration into the cerebellum, and significant neuroinflammation and paraneoplastic neurologic disorder developed only after anti-CTLA4 monoclonal antibody treatment. Moreover, our data strongly suggest that CD8 + T cells play a final effector role by killing the Purkinje neurons. Taken together, we recommend heightened caution when using CTLA4 blockade in patients with gynaecological cancers, or malignancies of neuroectodermal origin, such as small cell lung cancer, as such treatment may promote paraneoplastic neurologic disorders.

Neurons and T cells: Understanding this interaction for inflammatory neurological diseases.

Central nervous system (CNS) inflammation occurs in a large number of neurological diseases. The type and magnitude of CNS inflammation, as well as the T-cell contribution, vary depending on the disease. Different animal models of neurological diseases have shown that T cells play an important role in CNS inflammation. Furthermore, recent studies of human neurological disorders have indicated a significant role for T cells in disease pathology. Nevertheless, how individual T-cell subsets affect neuronal survival, damage and/or loss remains largely unclear. In this review we discuss the processes by which T cells mediate either beneficial or deleterious effects within the CNS, with emphasis on the direct interaction between T cells and neurons, as occurs in multiple sclerosis, paraneoplastic cerebellar degeneration, and viral encephalitis. The therapeutic approaches targeting T cells and their mediators as treatment for neurological diseases are also described here.

Migration of encephalitogenic CD8 T cells into the central nervous system is dependent on the α4ß1-integrin.

Although CD8 T cells are key players in neuroinflammation, little is known about their trafficking cues into the central nervous system (CNS). We used a murine model of CNS autoimmunity to define the molecules involved in cytotoxic CD8 T-cell migration into the CNS. Using a panel of mAbs, we here show that the α4ß1-integrin is essential for CD8 T-cell interaction with CNS endothelium. We also investigated which α4ß1-integrin ligands expressed by endothelial cells are implicated. The blockade of VCAM-1 did not protect against autoimmune encephalomyelitis, and only partly decreased the CD8(+) T-cell infiltration into the CNS. In addition, inhibition of junctional adhesion molecule-B expressed by CNS endothelial cells also decreases CD8 T-cell infiltration. CD8 T cells may use additional and possibly unidentified adhesion molecules to gain access to the CNS.

Suppression of MAPK attenuates neuronal cell death induced by activated glia-conditioned medium in alpha-synuclein overexpressing SH-SY5Y cells.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disease with characteristics and symptoms that are well defined. Nevertheless, its aetiology remains unknown. PD is characterized by the presence of Lewy bodies inside neurons. α-Synuclein (α-syn) is a soluble protein present in the pre-synaptic terminal of neurons. Evidence suggests that α-syn has a fundamental role in PD pathogenesis, given that it is an important component of Lewy bodies localized in the dopaminergic neurons of PD patients. METHODS: In the present study, we investigated the influence of wild type (WT) and A30P α-syn overexpression on neuroblastoma SH-SY5Y toxicity induced by the conditioned medium (CM) from primary cultures of glia challenged with lipopolysaccharide (LPS) from Escherichia coli. RESULTS: We observed that SH-SY5Y cells transduced with α-syn (WT or A30P) and treated with CM from LPS-activated glia cells show evidence of cell death, which is not reverted by NF-κB inhibition by sodium salicylate or by blockage of P50 (NF-κB subunit). Furthermore, the expression of A30P α-syn in neuroblastoma SH-SY5Y decreases the cell death triggered by the CM of activated glia versus WT α-syn or control group. This effect of A30P α-syn may be due to the low MAPK42/44 phosphorylation. This finding is substantiated by MEK1 inhibition by PD98059, decreasing LDH release by CM in SH-SY5Y cells. CONCLUSION: Our results suggest that SH-SY5Y cells transduced with α-syn (WT or A30P) and treated with CM from LPS-activated glia cells show cell death, which is not reverted by NF-κB blockage. Additionally, the expression of A30P α-syn on neuroblastoma SH-SY5Y leads to decreased cell death triggered by the CM of activated glia, when compared to WT α-syn or control group. The mechanism underlying this process remains to be completely elucidated, but the present data suggest that MAPK42/44 phosphorylation plays an important role in this process. PROSPERO: CRD42015020829.

Peripheral neurokinin-1 receptors contribute to kaolin-induced acute monoarthritis in rats.

OBJECTIVE: intra-articular co-injection of kaolin with carrageenan (CGN) in rodents is widely used as an experimental model of arthritis. However, the ability of kaolin to cause arthritis and related immune responses when administered alone is unclear. We evaluated the contribution of prostanoids and sensory C-fibres (and their neuropeptide substance P) to kaolin-induced inflammation in the rat knee. METHODS: Wistar rats, 8-10 weeks old, received an intra-articular injection of kaolin (1-10 µg/joint) or saline into the knee joint. Knee inflammation, proinflammatory cytokines, pain behaviour and secondary tactile allodynia were assessed over 5 h, when synovial leukocyte counts, histopathological changes and proinflammatory cytokine levels were evaluated. RESULTS: The intra-articular injection of kaolin caused a dose- and time-dependent knee swelling and impairment of motion that were associated with secondary tactile allodynia, elevated concentrations of IL-1ß, IL-6 and TNFα, leukocyte infiltration, and histopathological changes in the ipsilateral hindpaw. The neurokinin-1 (NK1) receptor antagonist SR140333 or neonatal treatment with capsaicin markedly reduced the inflammatory parameters, cytokines and allodynia but failed to significantly inhibit the impaired motion. The cyclo-oxygenase inhibitor indomethacin partially inhibited knee oedema and allodynia but did not affect the leukocyte influx, myeloperoxidase activity or impaired motion in the kaolin-injected rat. CONCLUSIONS: We show the first evidence that intra-articular injection of kaolin without CGN produced severe acute monoarthritis. This was highly dependent on substance P (released from C-fibres) and NK1 receptor activation, which stimulated local production of proinflammatory cytokines. This model may be of critical importance for mechanistic studies and screening new anti-inflammatory/analgesic drugs.

Intermittent fasting attenuates lipopolysaccharide-induced neuroinflammation and memory impairment.

BACKGROUND: Systemic bacterial infections often result in enduring cognitive impairment and are a risk factor for dementia. There are currently no effective treatments for infection-induced cognitive impairment. Previous studies have shown that intermittent fasting (IF) can increase the resistance of neurons to injury and disease by stimulating adaptive cellular stress responses. However, the impact of IF on the cognitive sequelae of systemic and brain inflammation is unknown. METHODS: Rats on IF for 30 days received 1 mg/kg of lipopolysaccharide (LPS) or saline intravenously. Half of the rats were subjected to behavioral tests and the other half were euthanized two hours after LPS administration and the hippocampus was dissected and frozen for analyses. RESULTS: Here, we report that IF ameliorates cognitive deficits in a rat model of sepsis by a mechanism involving NF-κB activation, suppression of the expression of pro-inflammatory cytokines, and enhancement of neurotrophic support. Treatment of rats with LPS resulted in deficits in cognitive performance in the Barnes maze and inhibitory avoidance tests, without changing locomotor activity, that were ameliorated in rats that had been maintained on the IF diet. IF also resulted in reduced levels of mRNAs encoding the LPS receptor TLR4 and inducible nitric oxide synthase (iNOS) in the hippocampus. Moreover, IF prevented LPS-induced elevation of IL-1α, IL-1ß and TNF-α levels, and prevented the LPS-induced reduction of BDNF levels in the hippocampus. IF also significantly attenuated LPS-induced elevations of serum IL-1ß, IFN-γ, RANTES, TNF-α and IL-6 levels. CONCLUSIONS: Taken together, our results suggest that IF induces adaptive responses in the brain and periphery that can suppress inflammation and preserve cognitive function in an animal model of systemic bacterial infection.

Signaling function of Na,K-ATPase induced by ouabain against LPS as an inflammation model in hippocampus.

BACKGROUND: Ouabain (OUA) is a newly recognized hormone that is synthesized in the adrenal cortex and hypothalamus. Low doses of OUA can activate a signaling pathway by interaction with Na,K-ATPase, which is protective against a number of insults. OUA has central and peripheral anti-inflammatory effects. Lipopolysaccharide (LPS), via toll-like receptor 4 activation, is a widely used model to induce systemic inflammation. This study used a low OUA dose to evaluate its effects on inflammation induced by LPS injection in rats. METHODS: Adult male Wistar rats received acute intraperitoneal (ip) OUA (1.8 µg/kg) or saline 20 minutes before LPS (200 µg/kg, ip) or saline injection. Some of the animals had their femoral artery catheterized in order to assess arterial blood pressure values before and after OUA administration. Na,K-ATPase activity, cytokine mRNA levels, apoptosis-related proteins, NF-κB activation brain-derived neurotrophic factor BDNF, corticosterone and TNF-α levels were measured. RESULTS: OUA pretreatment decreased mRNA levels of the pro-inflammatory cytokines, inducible nitric oxide synthase (iNOS) and IL-1ß, which are activated by LPS in the hippocampus, but with no effect on serum measures of these factors. None of these OUA effects were linked to Na,K-ATPase activity. The involvement of the inflammatory transcription factor NF-κB in the OUA effect was indicated by its prevention of LPS-induced nuclear translocation of the NF-κB subunit, RELA (p65), as well as the decreased cytosol levels of the NF-κB inhibitor, IKB, in the hippocampus. OUA pretreatment reversed the LPS-induced glial fibrillary acidic protein (GFAP) activation and associated inflammation in the dentate gyrus. OUA also prevented LPS-induced increases in the hippocampal Bax/Bcl2 ratio suggesting an anti-apoptotic action in the brain. CONCLUSION: Our results suggest that a low dose of OUA has an important anti-inflammatory effect in the rat hippocampus. This effect was associated with decreased GFAP induction by LPS in the dentate gyrus, a brain area linked to adult neurogenesis.

OTULIN haploinsufficiency predisposes to environmentally directed inflammation.

Recently, OTULIN haploinsufficiency was linked to enhanced susceptibility to Staphylococcus aureus infections accompanied by local necrosis and systemic inflammation. The pathogenesis observed in haploinsufficient patients differs from the hyperinflammation seen in classical OTULIN-related autoinflammatory syndrome (ORAS) patients and is characterized by increased susceptibility of dermal fibroblasts to S. aureus alpha toxin-inflicted cytotoxic damage. Immunological abnormalities were not observed in OTULIN haploinsufficient patients, suggesting a non-hematopoietic basis. In this research report, we investigated an Otulin+/- mouse model after in vivo provocation with lipopolysaccharide (LPS) to explore the potential role of hematopoietic-driven inflammation in OTULIN haploinsufficiency. We observed a hyperinflammatory signature in LPS-provoked Otulin+/- mice, which was driven by CD64+ monocytes and macrophages. Bone marrow-derived macrophages (BMDMs) of Otulin+/- mice demonstrated higher proinflammatory cytokine secretion after in vitro stimulation with LPS or polyinosinic:polycytidylic acid (Poly(I:C)). Our experiments in full and mixed bone marrow chimeric mice suggest that, in contrast to humans, the observed inflammation was mainly driven by the hematopoietic compartment with cell-extrinsic effects likely contributing to inflammatory outcomes. Using an OTULIN haploinsufficient mouse model, we validated the role of OTULIN in the regulation of environmentally directed inflammation.

Brain regulatory T cells.

The brain, long thought to be isolated from the peripheral immune system, is increasingly recognized to be integrated into a systemic immunological network. These conduits of immune-brain interaction and immunosurveillance processes necessitate the presence of complementary immunoregulatory mechanisms, of which brain regulatory T cells (Treg cells) are likely a key facet. Treg cells represent a dynamic population in the brain, with continual influx, specialization to a brain-residency phenotype and relatively rapid displacement by newly incoming cells. In addition to their functions in suppressing adaptive immunity, an emerging view is that Treg cells in the brain dampen down glial reactivity in response to a range of neurological insults, and directly assist in multiple regenerative and reparative processes during tissue pathology. The utility and malleability of the brain Treg cell population make it an attractive therapeutic target across the full spectrum of neurological conditions, ranging from neuroinflammatory to neurodegenerative and even psychiatric diseases. Therapeutic modalities currently under intense development include Treg cell therapy, IL-2 therapy to boost Treg cell numbers and multiple innovative approaches to couple these therapeutics to brain delivery mechanisms for enhanced potency. Here we review the state of the art of brain Treg cell knowledge together with the potential avenues for future integration into medical practice.

Molecular and cognitive signatures of ageing partially restored through synthetic delivery of IL2 to the brain.

Cognitive decline is a common pathological outcome during aging, with an ill-defined molecular and cellular basis. In recent years, the concept of inflammaging, defined as a low-grade inflammation increasing with age, has emerged. Infiltrating T cells accumulate in the brain with age and may contribute to the amplification of inflammatory cascades and disruptions to the neurogenic niche observed with age. Recently, a small resident population of regulatory T cells has been identified in the brain, and the capacity of IL2-mediated expansion of this population to counter neuroinflammatory disease has been demonstrated. Here, we test a brain-specific IL2 delivery system for the prevention of neurological decline in aging mice. We identify the molecular hallmarks of aging in the brain glial compartments and identify partial restoration of this signature through IL2 treatment. At a behavioral level, brain IL2 delivery prevented the age-induced defect in spatial learning, without improving the general decline in motor skill or arousal. These results identify immune modulation as a potential path to preserving cognitive function for healthy aging.

Brain-resident regulatory T cells and their role in health and disease.

Regulatory T cells (Tregs) control inflammation and maintain immune homeostasis. The well-characterised circulatory population of CD4+Foxp3+ Tregs is effective at preventing autoimmunity and constraining the immune response, through direct and indirect restraint of conventional T cell activation. Recent advances in Treg cell biology have identified tissue-resident Tregs, with tissue-specific functions that contribute to the maintenance of tissue homeostasis and repair. A population of brain-resident Tregs, characterised as CD69+, has recently been identified in the healthy brain of mice and humans, with rapid population expansion observed under a number of neuroinflammatory conditions. During neuroinflammation, brain-resident Tregs have been proposed to control astrogliosis through the production of amphiregulin, polarize microglia into neuroprotective states, and restrain inflammatory responses by releasing IL-10. While protective effects for Tregs have been demonstrated in a number of neuroinflammatory pathologies, a clear demarcation between the role of circulatory and brain-resident Tregs has been difficult to achieve. Here we review the state-of-the-art for brain-resident Treg population, and describe their potential utilization as a therapeutic target across different neuroinflammatory conditions.

Intratumoral DNA-based delivery of checkpoint-inhibiting antibodies and interleukin 12 triggers T cell infiltration and anti-tumor response.

To improve the anti-tumor efficacy of immune checkpoint inhibitors, numerous combination therapies are under clinical evaluation, including with IL-12 gene therapy. The current study evaluated the simultaneous delivery of the cytokine and checkpoint-inhibiting antibodies by intratumoral DNA electroporation in mice. In the MC38 tumor model, combined administration of plasmids encoding IL-12 and an anti-PD-1 antibody induced significant anti-tumor responses, yet similar to the monotherapies. When treatment was expanded with a DNA-based anti-CTLA-4 antibody, this triple combination significantly delayed tumor growth compared to IL-12 alone and the combination of anti-PD-1 and anti-CTLA-4 antibodies. Despite low drug plasma concentrations, the triple combination enabled significant abscopal effects in contralateral tumors, which was not the case for the other treatments. The DNA-based immunotherapies increased T cell infiltration in electroporated tumors, especially of CD8+ T cells, and upregulated the expression of CD8+ effector markers. No general immune activation was detected in spleens following either intratumoral treatment. In B16F10 tumors, evaluation of the triple combination was hampered by a high sensitivity to control plasmids. In conclusion, intratumoral gene electrotransfer allowed effective combined delivery of multiple immunotherapeutics. This approach induced responses in treated and contralateral tumors, while limiting systemic drug exposure and potentially detrimental systemic immunological effects.