Calcium/calmodulin-dependent protein kinase kinase 2 regulates macrophage-mediated inflammatory responses.

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) plays a key role in regulating food intake and energy expenditure at least in part by its actions in hypothalamic neurons. Previously, we showed that loss of CaMKK2 protected mice from high-fat diet (HFD)-induced obesity and glucose intolerance. However, although pair feeding HFD to WT mice to match food consumption of CAMKK2-null mice slowed weight gain, it failed to protect from glucose intolerance. Here we show that relative to WT mice, HFD-fed CaMKK2-null mice are protected from inflammation in adipose and remain glucose-tolerant. Moreover, loss of CaMKK2 also protected mice from endotoxin shock and fulminant hepatitis. We explored the expression of CaMKK2 in immune cells and found it to be restricted to those of the monocyte/macrophage lineage. CaMKK2-null macrophages exhibited a remarkable deficiency to spread, phagocytose bacteria, and synthesize cytokines in response to the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS). Mechanistically, loss of CaMKK2 uncoupled the TLR4 cascade from activation of protein tyrosine kinase 2 (PYK2; also known as PTK2B). Our findings uncover an important function for CaMKK2 in mediating mechanisms that control the amplitude of macrophage inflammatory responses to excess nutrients or pathogen derivatives.

Differential diagnosis of mild cognitive impairment of Alzheimer's disease by Simoa p-tau181 measurements with matching plasma and CSF.

Alzheimer's disease (AD) is characterized by a long preclinical phase. Although late-stage AD/dementia may be robustly differentiated from cognitively normal individuals by means of a clinical evaluation, PET imaging, and established biofluid biomarkers, disease differentiation between cognitively normal and various subtypes of mild cognitive impairment (MCI) remains a challenging task. Differential biomarkers for early-stage AD diagnosis with accessible biofluid samples are urgently needed. Misfolded phosphorylated tau aggregates (p-tau) are present in multiple neurodegenerative diseases known as "tauopathies", with the most common being AD. P-tau181 is a well-established p-tau biomarker to differentiate AD dementia from non-AD pathology. However, it is unclear if p-tau181 is capable of diagnosing MCI, an early AD stage, from cognitively normal subjects, or if it can discriminate MCI subtypes amnestic MCI (aMCI) from non-amnestic MCI (naMCI). Here we evaluated the capability of p-tau181 in diagnosing MCI from cognitively normal subjects and discriminating aMCI from naMCI subtypes. We collected matching plasma and CSF samples of a clinically diagnosed cohort of 35 cognitively normal, 34 aMCI, 17 naMCI, and 31 AD dementia cases (total 117 participants) with supplemental CSF Aß42 and total tau AD biomarker levels and performed Simoa p-tau181 assays. The diagnostic capabilities of Simoa p-tau181 assays to differentiate these cohorts were evaluated. We found (i) p-tau181 can robustly differentiate MCI or aMCI from cognitively normal cohorts with matching plasma and CSF samples, but such differentiation is weaker in diagnosing naMCI from cognitively normal groups, (ii) p-tau181 is not capable of differentiating aMCI from naMCI cohorts, and (iii) either factor of Aß or total tau burden markedly improved differentiation power to diagnose aMCI from cognitively normal group. Plasma and CSF p-tau181 levels may serve as a promising biomarker for diagnosing aMCI from normal controls in the preclinical phase. But more robust new biomarkers are needed to differentiate naMCI from cognitively normal cases or to discriminate between MCI subtypes, aMCI from naMCI.

DAMPs/PAMPs induce monocytic TLR activation and tolerance in COVID-19 patients; nucleic acid binding scavengers can counteract such TLR agonists.

Millions of COVID-19 patients have succumbed to respiratory and systemic inflammation. Hyperstimulation of toll-like receptor (TLR) signaling is a key driver of immunopathology following infection by viruses. We found that severely ill COVID-19 patients in the Intensive Care Unit (ICU) display hallmarks of such hyper-stimulation with abundant agonists of nucleic acid-sensing TLRs present in their blood and lungs. These nucleic acid-containing Damage and Pathogen Associated Molecular Patterns (DAMPs/PAMPs) can be depleted using nucleic acid-binding microfibers to limit the patient samples' ability to hyperactivate such innate immune receptors. Single-cell RNA-sequencing revealed that CD16+ monocytes from deceased but not recovered ICU patients exhibit a TLR-tolerant phenotype and a deficient anti-viral response after ex vivo TLR stimulation. Plasma proteomics confirmed such myeloid hyperactivation and revealed DAMP/PAMP carrier consumption in deceased patients. Treatment of these COVID-19 patient samples with MnO nanoparticles effectively neutralizes TLR activation by the abundant nucleic acid-containing DAMPs/PAMPs present in their lungs and blood. Finally, MnO nanoscavenger treatment limits the ability of DAMPs/PAMPs to induce TLR tolerance in monocytes. Thus, treatment with microfiber- or nanoparticle-based DAMP/PAMP scavengers may prove useful for limiting SARS-CoV-2 induced hyperinflammation, preventing monocytic TLR tolerance, and improving outcomes in severely ill COVID-19 patients.

Profiling serum neurofilament light chain and glial fibrillary acidic protein in primary progressive multiple sclerosis.

This study examined the utility of serum neurofilament light chain (sNfL) and glial fibrillary acidic protein (sGFAP) as biomarkers in primary progressive multiple sclerosis in context with clinical severity, progression, and treatment. Using a single-molecule array (Quanterix), serum protein concentrations were measured from twenty-five participants semiannually for five years. There was no association between levels of either biomarker and disease severity, disease duration, or treatment group. Enrollment sNfL level was not associated with future clinical worsening. Precedent clinical worsening was not associated with last sGFAP measurement. These results suggest a limited role for these biomarkers in primary progressive disease management.

Calcium/calmodulin-dependent kinase kinase 2 regulates hematopoietic stem and progenitor cell regeneration.

Hematopoietic stem and progenitor cells (HSPCs) are predominantly quiescent in adults, but proliferate in response to bone marrow (BM) injury. Here, we show that deletion of Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) promotes HSPC regeneration and hematopoietic recovery following radiation injury. Using Camkk2-enhanced green fluorescent protein (EGFP) reporter mice, we found that Camkk2 expression is developmentally regulated in HSPC. Deletion of Camkk2 in HSPC results in a significant downregulation of genes affiliated with the quiescent signature. Accordingly, HSPC from Camkk2 null mice have a high proliferative capability when stimulated in vitro in the presence of BM-derived endothelial cells. In addition, Camkk2 null mice are more resistant to radiation injury and show accelerated hematopoietic recovery, enhanced HSPC regeneration and ultimately a prolonged survival following sublethal or lethal total body irradiation. Mechanistically, we propose that CaMKK2 regulates the HSPC response to hematopoietic damage by coupling radiation signaling to activation of the anti-proliferative AMP-activated protein kinase. Finally, we demonstrated that systemic administration of the small molecule CaMKK2 inhibitor, STO-609, to irradiated mice enhanced HSPC recovery and improved survival. These findings identify CaMKK2 as an important regulator of HSPC regeneration and demonstrate CaMKK2 inhibition is a novel approach to promoting hematopoietic recovery after BM injury.