Tregs Attenuate Peripheral Oxidative Stress and Acute Phase Proteins in ALS.

Oxidative stress (OS) induces inflammation, which in turn exacerbates OS and the expression of acute phase proteins (APPs). Regulatory T lymphocyte (Treg) therapy was assessed for suppression of OS and APP responses in longitudinal serum samples from subjects with amyotrophic lateral sclerosis (ALS) enrolled in a phase I clinical trial. The first round of Treg therapy suppressed levels of oxidized low-density lipoprotein (ox-LDL). During a 6-month washout period, ox-LDL levels increased. A second round of therapy again suppressed ox-LDL levels and then rose following the cessation of treatment. Serum levels of APPs, soluble CD14, lipopolysaccharide binding protein, and C-reactive protein, were stabilized during Treg administrations, but rose during the washout period and again after therapy was discontinued. Treg therapy potentially suppresses peripheral OS and the accompanying circulating pro-inflammatory induced APPs, both of which may serve as peripheral candidates for monitoring efficacies of immunomodulating therapies. ANN NEUROL 2022;92:195-200.

A phase 1 proof-of-concept study evaluating safety, tolerability, and biological marker responses with combination therapy of CTLA4-Ig and interleukin-2 in amyotrophic lateral sclerosis.

Objective: To determine whether a combination therapy with abatacept (CTLA4-Ig) and interleukin-2 (IL-2) is safe and suppresses markers of oxidative stress, inflammation, and degeneration in ALS. Methods: In this open-label study, four participants with ALS received subcutaneous injections of low dose IL-2 (1 × 106 IU/injection/day) for 5 consecutive days every 2 weeks and one subcutaneous injection of CTLA4-Ig (125 mg/mL/injection) every 2 weeks coinciding with the first IL-2 injection of each treatment cycle. Participants received a total of 24 treatment cycles during the first 48 weeks in this 56-week study. They were closely monitored for treatment-emergent adverse events (TEAEs) and disease progression with the ALSFRS-R. Phenotypic changes within T cell populations and serum biological markers of oxidative stress [4-hydroxynonenal (4-HNE) and oxidized-LDL (ox-LDL)], inflammation (IL-18), and structural neuronal degeneration [neurofilament light chain (Nf-L)] were assessed longitudinally. Results: CTLA4-Ig/IL-2 therapy was safe and well-tolerated in all four participants over the 56-week study. During the first 24 weeks, the average rate of change in the ALSFRS-R was +0.04 points/month. Over the 48-week treatment period, the average rate of change was -0.13 points/month with one participant improving by 0.9 points/month while the other three participants experienced an average decrease of -0.47 points/month, which is slower than the average - 1.1 points/month prior to initiation of therapy. Treg suppressive function and numbers increased during treatment. Responses in the biological markers during the first 16 weeks coincided with minimal clinical progression. Mean levels of 4-HNE decreased by 30%, ox-LDL decreased by 19%, IL-18 decreased by 23%, and Nf-L remained the same, on average, in all four participants. Oxidized-LDL levels decreased in all four participants, 4-HNE and IL-18 levels decreased in three out of four participants, and Nf-L decreased in two out of four participants. Conclusion: The combination therapy of CTLA4-Ig and IL-2 in ALS is safe and well-tolerated with promising results of clinical efficacy and suppression of biomarkers of oxidative stress, neuroinflammation and neuronal degeneration. In this open-label study, the efficacy as measured by the ALSFRS-R and corresponding biomarkers suggests the therapeutic potential of this treatment and warrants further study in a phase 2 double-blind, placebo-controlled trial. Clinical trial registration: ClinicalTrials.gov, NCT06307301.

Endogenous regulatory T lymphocytes ameliorate amyotrophic lateral sclerosis in mice and correlate with disease progression in patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a relentless and devastating adult-onset neurodegenerative disease with no known cure. In mice with amyotrophic lateral sclerosis, CD4+ T lymphocytes and wild-type microglia potentiate protective inflammatory responses and play a principal role in disease pathoprogression. Using this model, we demonstrate that endogenous T lymphocytes, and more specifically regulatory T lymphocytes, are increased at early slowly progressing stages, augmenting interleukin-4 expression and protective M2 microglia, and are decreased when the disease rapidly accelerates, possibly through the loss of FoxP3 expression in the regulatory T lymphocytes. Without ex vivo activation, the passive transfer of wild-type CD4+ T lymphocytes into amyotrophic lateral sclerosis mice lacking functional T lymphocytes lengthened disease duration and prolonged survival. The passive transfer of endogenous regulatory T lymphocytes from early disease stage mutant Cu2+/Zn2+ superoxide dismutase mice into these amyotrophic lateral sclerosis mice, again without ex vivo activation, were substantially more immunotherapeutic sustaining interleukin-4 levels and M2 microglia, and resulting in lengthened disease duration and prolonged survival; the stable disease phase was extended by 88% using mutant Cu2+/Zn2+ superoxide dismutase regulatory T lymphocytes. A potential mechanism for this enhanced life expectancy may be mediated by the augmented secretion of interleukin-4 from mutant Cu2+/Zn2+ superoxide dismutase regulatory T lymphocytes that directly suppressed the toxic properties of microglia; flow cytometric analyses determined that CD4+/CD25+/FoxP3+ T lymphocytes co-expressed interleukin-4 in the same cell. These observations were extended into the amyotrophic lateral sclerosis patient population where patients with more rapidly progressing disease had decreased numbers of regulatory T lymphocytes; the numbers of regulatory T lymphocytes were inversely correlated with disease progression rates. These data suggest a cellular mechanism whereby endogenous regulatory T lymphocytes are immunocompetent and actively contribute to neuroprotection through their interactions with microglia. Furthermore, these data suggest that immunotherapeutic interventions must begin early in the pathogenic process since immune dysfunction occurs at later stages. Thus, the cumulative mouse and human amyotrophic lateral sclerosis data suggest that increasing the levels of regulatory T lymphocytes in patients with amyotrophic lateral sclerosis at early stages in the disease process may be of therapeutic value, and slow the rate of disease progression and stabilize patients for longer periods of time.

Ex vivo expanded human regulatory T cells modify neuroinflammation in a preclinical model of Alzheimer's disease.

BACKGROUND: Regulatory T cells (Tregs) play a neuroprotective role by suppressing microglia and macrophage-mediated inflammation and modulating adaptive immune reactions. We previously documented that Treg immunomodulatory mechanisms are compromised in Alzheimer's disease (AD). Ex vivo expansion of Tregs restores and amplifies their immunosuppressive functions in vitro. A key question is whether adoptive transfer of ex vivo expanded human Tregs can suppress neuroinflammation and amyloid pathology in a preclinical mouse model. METHODS: An immunodeficient mouse model of AD was generated by backcrossing the 5xFAD onto Rag2 knockout mice (5xFAD-Rag2KO). Human Tregs were expanded ex vivo for 24 days and administered to 5xFAD-Rag2KO. Changes in amyloid burden, microglia characteristics and reactive astrocytes were evaluated using ELISA and confocal microscopy. NanoString Mouse AD multiplex gene expression analysis was applied to explore the impact of ex vivo expanded Tregs on the neuroinflammation transcriptome. RESULTS: Elimination of mature B and T lymphocytes and natural killer cells in 5xFAD-Rag2KO mice was associated with upregulation of 95 inflammation genes and amplified number of reactive microglia within the dentate gyrus. Administration of ex vivo expanded Tregs reduced amyloid burden and reactive glial cells in the dentate gyrus and frontal cortex of 5xFAD-Rag2KO mice. Interrogation of inflammation gene expression documented down-regulation of pro-inflammatory cytokines (IL1A&B, IL6), complement cascade (C1qa, C1qb, C1qc, C4a/b), toll-like receptors (Tlr3, Tlr4 and Tlr7) and microglial activations markers (CD14, Tyrobp,Trem2) following Treg administration. CONCLUSIONS: Ex vivo expanded Tregs with amplified immunomodulatory function, suppressed neuroinflammation and alleviated AD pathology in vivo. Our results provide preclinical evidences for Treg cell therapy as a potential treatment strategy in AD.

Serum programmed cell death proteins in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a multifactorial, multisystem pro-inflammatory neuromuscular disorder. Activation of programmed cell death-1 (PD-1), and its ligands, programmed cell death-ligand 1 and 2 (PD-L1/L2), leads to immune suppression. Serum soluble forms of these proteins, sPD-1/sPD-L1/sPD-L2, inhibit this suppression and promote pro-inflammatory responses. The purpose of this study was to determine if sPD-1, sPD-L1, and sPD-L2 were increased in sera of patients with ALS. sPD-1 and sPD-L2 were elevated in sera of patients and accurately reflected patients' disease burdens. Increased sera levels of programmed cell death proteins reinforce the concept that peripheral pro-inflammatory responses contribute to systemic inflammation in patients with ALS.

Increased activation ability of monocytes from ALS patients.

Neuroinflammation is increasingly recognized as an important mediator of disease progression in patients with amyotrophic lateral sclerosis (ALS). Recent research suggests that pro-inflammatory microglia in ALS mice promote motoneuron cytotoxicity by secreting reactive oxygen species and pro-inflammatory cytokines. Gene expression analyses indicate that peripheral circulating monocytes from ALS patients are skewed towards a pro-inflammatory state that contributes to ALS disease progression. Better understanding of macrophage phenotypes of ALS patients is therefore warranted. In this study, we demonstrate that M1 macrophages differentiated from ALS circulating monocytes produced more pro-inflammatory cytokines, including IL-6 and TNFα, than M1 macrophages derived from healthy control monocytes. More importantly, IL-6 protein levels of ALS M1 macrophages positively correlated with disease burden, and TNFα protein levels of ALS M1 macrophages positively correlate with disease progression rates. Collectively, these data suggest that monocytes from ALS patients are more readily activated and differentiated to a pro-inflammatory M1 phenotype, and represent a potential target for immunomodulatory therapy.

Elevated acute phase proteins reflect peripheral inflammation and disease severity in patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a multifactorial, multisystem pro-inflammatory neuromuscular disorder compromising muscle function resulting in death. Neuroinflammation is known to accelerate disease progression and accentuate disease severity, but peripheral inflammatory processes are not well documented. Acute phase proteins (APPs), plasma proteins synthesized in the liver, are increased in response to inflammation. The objective of this study was to provide evidence for peripheral inflammation by examining levels of APPs, and their contribution to disease burden and progression rates. Levels of APPs, including soluble CD14 (sCD14), lipopolysaccharide binding protein (LBP), and C-reactive protein (CRP), were elevated in sera, and correlated positively with increased disease burden and faster progression. sCD14 was also elevated in patients' CSF and urine. After a 3 year follow-up, 72% of the patients with sCD14 levels above the receiver operating characteristics cutoff were deceased whereas only 28% below the cutoff were deceased. Furthermore, disease onset sites were associated with disease progression rates and APP levels. These APPs were not elevated in sera of patients with Alzheimer's Disease, frontotemporal dementia, or Parkinson's Disease. These collective APPs accurately reflect disease burden, progression rates, and survival times, reinforcing the concept of ALS as a disorder with extensive systemic pro-inflammatory responses.

Immunosuppressive Functions of M2 Macrophages Derived from iPSCs of Patients with ALS and Healthy Controls.

Amyotrophic lateral sclerosis (ALS) is a disorder with immune alterations that augment disease severity. M2 macrophages benefit diabetic and nephrotic mice by suppressing the pro-inflammatory state. However, neither have M2 cells been investigated in ALS nor have human induced pluripotent stem cell (iPSC)-derived M2 cells been thoroughly studied for immunosuppressive potentials. Here, iPSCs of C9orf72 mutated or sporadic ALS patients were differentiated into M2 macrophages, which suppressed activation of pro-inflammatory M1 macrophages as well as proliferation of ALS CD4+CD25- Tc (Teffs). M2 cells converted ALS Teffs into CD4+CD25+Foxp3+ regulatory T cells (Tregs) and rescued Tregs of ALS patients from losing CD25 and Foxp3. Furthermore, Tregs induced or rescued by iPSC-derived M2 had strong suppressive functions. ALS iPSC-derived M2 cells including those with C9orf72 mutation had similar immunomodulatory activity as control iPSC-derived M2 cells. This study demonstrates that M2 cells differentiated from iPSCs of ALS patients are immunosuppressive, boost ALS Tregs, and may serve as a candidate for immune-cell-based therapy to mitigate inflammation in ALS.

Functional alterations of myeloid cells during the course of Alzheimer's disease.

BACKGROUND: Neuroinflammation is a hallmark of neurodegenerative disease and a significant component of the pathology of Alzheimer's disease (AD). Patients present with extensive microgliosis along with elevated pro-inflammatory signaling in the central nervous system and periphery. However, the role of peripheral myeloid cells in mediating and influencing AD pathogenesis remains unresolved. METHODS: Peripheral myeloid cells were isolated from peripheral blood of patients with prodromal AD (n = 44), mild AD dementia (n = 25), moderate/severe AD dementia (n = 28), and age-matched controls (n = 54). Patients were evaluated in the clinic for AD severity and categorized using Clinical Dementia Rating (CDR) scale resulting in separation of patients into prodromal AD (CDR0.5) and advancing forms of AD dementia (mild-CDR1 and moderate/severe-CDR2/3). Separation of peripheral myeloid cells into mature monocytes or immature MDSCs permitted the delineation of population changes from flow cytometric analysis, RNA phenotype analysis, and functional studies using T cell suppression assays and monocyte suppression assays. RESULTS: During stages of AD dementia (CDR1 and 2/3) peripheral myeloid cells increase their pro-inflammatory gene expression while at early stages of disease (prodromal AD-CDR0.5) pro-inflammatory gene expression is decreased. MDSCs are increased in prodromal AD compared with controls (16.81% vs 9.53%) and have markedly increased suppressive functions: 42.4% suppression of activated monocyte-produced IL-6 and 78.16% suppression of T cell proliferation. In AD dementia, MDSC populations are reduced with decreased suppression of monocyte IL-6 (5.22%) and T cell proliferation (37.61%); the reduced suppression coincides with increased pro-inflammatory signaling in AD dementia monocytes. CONCLUSIONS: Peripheral monocyte gene expression is pro-inflammatory throughout the course of AD, except at the earliest, prodromal stages when pro-inflammatory gene expression is suppressed. This monocyte biphasic response is associated with increased numbers and suppressive functions of MDSCs during the early stages and decreased numbers and suppressive functions in later stages of disease. Prolonging the early protective suppression and reversing the later loss of suppressive activity may offer a novel therapeutic strategy.

ALS patients' regulatory T lymphocytes are dysfunctional, and correlate with disease progression rate and severity.

Neuroinflammation is a pathological hallmark of ALS in both transgenic rodent models and patients, and is characterized by proinflammatory T lymphocytes and activated macrophages/microglia. In ALS mouse models, decreased regulatory T lymphocytes (Tregs) exacerbate the neuroinflammatory process, leading to accelerated motoneuron death and shortened survival; passive transfer of Tregs suppresses the neuroinflammation and prolongs survival. Treg numbers and FOXP3 expression are also decreased in rapidly progressing ALS patients. A key question is whether the marked neuroinflammation in ALS can be attributed to the impaired suppressive function of ALS Tregs in addition to their decreased numbers. To address this question, T lymphocyte proliferation assays were performed. Compared with control Tregs, ALS Tregs were less effective in suppressing responder T lymphocyte proliferation. Although both slowly and rapidly progressing ALS patients had dysfunctional Tregs, the greater the clinically assessed disease burden or the more rapidly progressing the patient, the greater the Treg dysfunction. Epigenetically, the percentage methylation of the Treg-specific demethylated region was greater in ALS Tregs. After in vitro expansion, ALS Tregs regained suppressive abilities to the levels of control Tregs, suggesting that autologous passive transfer of expanded Tregs might offer a novel cellular therapy to slow disease progression.

TDP-43 activates microglia through NF-κB and NLRP3 inflammasome.

Transactive response DNA-binding protein-43 (TDP-43) is a multifunctional nucleic acid binding protein present in ubiquitinated inclusions in tissues of patients with amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD). The ALS-associated mutations in the glycine-rich C-terminal domain of TDP-43 established a causal link between TDP-43 and disease, and conferred both loss- and gain-of-function properties in neurons. Since it has not been established whether these intra-neuronal changes are sufficient to cause ALS or whether non-cell autonomous neuronal-glial signaling could be involved, we investigated the extracellular effects of TDP-43 proteins on microglial activation and motoneuron toxicity. Wild-type, truncated 25kD C-terminal fragments, or mutant forms of TDP-43 all activated microglia and upregulated NOX2, TNF-α, and IL-1ß, with WT forms being significantly less effective in activating microglia. This response to TDP-43 was mediated by its interaction with the microglial surface CD14 receptor and subsequent stimulation of the NF-κB and AP-1 pathways, as well as the intracellular inflammasome. At the cell surface, CD14 blocking antibodies suppressed microglial NF-κB activation and proinflammatory cytokine production mediated by TDP-43. Intracellularly, the NLRP3 inflammasome was induced and functional caspase-1 was produced augmenting the release of mature IL-1ß. Further, TDP-43-mediated activation of microglia caused a proinflammatory cascade that was toxic to motoneurons. In the absence of microglia, TDP-43 was not toxic to motoneurons. The ability of TDP-43 to promote CD14-mediated activation of microglial NF-κB and AP-1 pathways, as well as the NLRP3 inflammasome, suggests the involvement of a non-cell autonomous proinflammatory signaling that enhances motoneuron injury, and may offer novel therapeutic targets in ALS.

Deployment of balloon expandable stents during open repair of thoracoabdominal aortic aneurysms: a new strategy for managing renal and mesenteric artery lesions.

OBJECTIVE: Patients undergoing graft repair of thoracoabdominal aortic aneurysms (TAAAs) often require concomitant correction of ostial stenoses or dissection involving visceral branches. The purpose of this report is to describe our initial experience with a new strategy for addressing these lesions during open TAAA repair-direct deployment of balloon expandable stents into the renal and mesenteric arteries. METHODS: Over a two-year period, 367 patients have undergone TAAA surgery. Balloon expandable stents were used to manage visceral branch lesions during open TAAA repair in 93 (25.3%) of these patients. Fifteen patients (16%) had preoperative renal insufficiency. After opening the aneurysm and exposing the branch artery ostia, premounted balloon expandable stents were deployed in the affected vessels under direct vision. Stents were deployed after an endarterectomy in 40 patients (43%). Eighty patients (86%) had stents placed in one or both renal arteries and 36 (39%) had stents placed in the celiac axis and/or superior mesenteric artery. Postoperative renal function was monitored with daily serum creatinine levels. RESULTS: There were nine early operative deaths (10%). Two patients (2%) had bleeding complications related to stenting, one of which died after developing multiple organ failure. Twelve patients (13%) developed renal failure, eight of which required dialysis. CONCLUSIONS: This study demonstrates the feasibility of treating ostial lesions of the visceral branches with balloon expandable stents during open TAAA repair. Despite a high prevalence of preoperative renal insufficiency, the incidence of postoperative renal failure was acceptable. This new strategy may be a valuable adjunct to TAAA repair and warrants further investigation.

Regulatory T-lymphocytes mediate amyotrophic lateral sclerosis progression and survival.

In amyotrophic lateral sclerosis (ALS) mice, regulatory T-lymphocytes (Tregs) are neuroprotective, slowing disease progression. To address whether Tregs and FoxP3, a transcription factor required for Treg function, similarly influence progression rates of ALS patients, T-lymphocytes from patients were assessed by flow cytometry. Both numbers of Tregs and their FoxP3 protein expressions were reduced in rapidly progressing ALS patients and inversely correlated with progression rates. The mRNA levels of FoxP3, TGF-ß, IL4 and Gata3, a Th2 transcription factor, were reduced in rapidly progressing patients and inversely correlated with progression rates. Both FoxP3 and Gata3 were accurate indicators of progression rates. No differences in IL10, Tbx21, a Th1 transcription factor or IFN-γ expression were found between slow and rapidly progressing patients. A 3.5-year prospective study with a second larger cohort revealed that early reduced FoxP3 levels were indicative of progression rates at collection and predictive of future rapid progression and attenuated survival. Collectively, these data suggest that Tregs and Th2 lymphocytes influence disease progression rates. Importantly, early reduced FoxP3 levels could be used to identify rapidly progressing patients.

Matrix metalloproteinases in ascending aortic aneurysms: bicuspid versus trileaflet aortic valves.

BACKGROUND: Abnormal matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) expression contributes to the development of abdominal aortic aneurysms. Recent data suggest that MMP-2 and MMP-9 may also play a role in thoracic aortic disease. We sought to determine (1) whether ascending aortic aneurysms are associated with increased MMP expression and (2) whether aortic inflammation and MMP expression differ between patients with congenital bicuspid aortic valves (BAVs) and those with trileaflet aortic valves (TAVs). MATERIALS AND METHODS: Samples of ascending aortic aneurysms were obtained from 29 patients; 14 patients had BAVs and 15 had TAVs. Control ascending aorta was obtained from 14 organ donors or heart transplant recipients. Aortic histology and immunohistochemistry were performed to evaluate elastin degradation, inflammatory changes, and MMP-2 and MMP-9 expression. Aortic levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 were measured using ELISA. RESULTS: Aneurysms in the TAV patients exhibited marked inflammation, high CD68 expression, diminished elastin content, increased MMP-9 expression, and normal MMP-2 levels. In contrast, BAV aneurysms were characterized by a relative lack of inflammation, preservation of elastin content, normal MMP-9 levels, and elevated MMP-2 expression. TIMP-1 and TIMP-2 levels were not significantly different among the three groups. CONCLUSIONS: Ascending aortic aneurysms exhibited increased MMP expression. The pattern of MMP expression and the degree of inflammation, however, differed between aneurysms associated with BAVs and those with TAVs. Variations in the molecular mechanisms underlying different types of thoracic aortic aneurysms warrant further investigation.