AA-Amyloidosis Can Be Transferred by Peripheral Blood Monocytes

Spongiform encephalopathies have been reported to be transmitted by blood transfusion even prior to the clinical onset. Experimental AA-amyloidosis shows similarities with prion disease and amyloid-containing organ-extracts can prime a recipient for the disease. In this systemic form of amyloidosis N-terminal fragments of the acute-phase reactant apolipoprotein serum amyloid A are the main amyloid protein. Initial amyloid deposits appear in the perifollicular region of the spleen, followed by deposits in the liver. We used the established murine model and induced AA-amyloidosis in NMRI mice by intravenous injections of purified amyloid fibrils (‘amyloid enhancing factor’) combined with inflammatory challenge (silver nitrate subcutaneously). Blood plasma and peripheral blood monocytes were isolated, sonicated and re-injected into new recipients followed by an inflammatory challenge during a three week period. When the animals were sacrificed presence of amyloid was analyzed in spleen sections after Congo red staining. Our result shows that some of the peripheral blood monocytes, isolated from animals with detectable amyloid, contained amyloid-seed that primed for AA-amyloid. The seeding material seems to have been phagocytosed by the cells since the AA-precursor (SAA1) was found not be expressed by the monocytes. Plasma recovered from mice with AA amyloidosis lacked seeding capacity. Amyloid enhancing activity can reside in monocytes recovered from mice with AA-amyloidosis and in a prion-like way trigger amyloid formation in conjunction with an inflammatory disorder. Human AA-amyloidosis resembles the murine form and every individual is expected to be exposed to conditions that initiate production of the acute-phase reactant. The monocyte-transfer mechanism should be eligible for the human disease and we point out blood transfusion as a putative route for transfer of amyloidosis

Analysis of AA/SAA reactivity in peripheral blood monocytes by confocal microscopy showed immunoreactivity in 5% of the monocytes isolated from a mouse with AA-amyloidosis (A).

<p>There was no reactivity present in monocytes recovered from a mouse given one AgNO₃ injection 48 hrs prior to isolation (B) or in monocytes isolated from untreated mice (C). The used rabbit antiserum recognizes both protein AA and SAA and was visualized by goat anti rabbit Alexa488-cojugated IgG. Cell nuclei were labeled with TO-PRO3. Bar 10 um.</p

Analysis of AEF activity in peripheral blood monocytes isolated from mice with AA-amyloid induced by AEF.

<p>AA-amyloid was induced in nine mice (G1–G9) by an i.v. injection of 0.1 ml AEF with concomitant s.c. injection of 0.2 ml 1% silver nitrate day 1, 7, 14, 21 and 28 and the mice were sacrificed on day 35. The presence of amyloid in the spleen was verified by Congo red staining. Peripheral blood monocytes were isolated, sonicated and re-introduced into the blood circulation of new groups of healthy mice (H1–H8). These mice received inflammatory stimuli day 1, 7 and 14 and were sacrificed day 16. The presence of amyloid was analysed in spleen sections after Congo red staining. Mice in group H9 received sonicated monocytes without subsequent inflammatory stimuli and group H10 received monocytes isolated from untreated mice and subsequent inflammatory stimuli on day 1, 7 and 14 and were sacrificed day 16 (group H10).</p

Analysis of AEF activity in peripheral blood monocytes isolated from mice with AA amyloidosis.

<p>The table presents detailed information on animals in group H 1–8. AA-amyloidosis was induced by i.v. injection of AEF and 0.2 ml 1% silver nitrate injections on day 1, 7, 14, 21 and 28. The animals were sacrificed on day 35. Blood was collected and amyloid was verified in spleen sections after Congo red staining. Isolated peripheral blood monocytes were injected into new animals, group H 1–8, and silver nitrate was given day 1, 7 and 14.The animals were sacrificed day 16 and the presence of amyloid was studied in spleen after Congo red staining.</p

SAA 1, SAA 2 and SAA 3 mRNA expression in peripheral blood monocytes were analysed with PCR.

<p>Cells were isolated from mice that developed AA-amyloid after AEF and AgNO₃ injections or from mice that received AEF or AgNO₃ injections only or from untreated mice. Expression of the amyloid-prone SAA 1 or non-amyloidogenic SAA 2 was absent in all monocyte preparations. SAA 3 mRNA was detected in all cells independent of treatment. Mouse liver cDNA was used as a positive control. The PCR products were separated on a 1.6% agarose gel.</p

Spleen amyloid deposits stained with Congo red.

<p>(A) The amyloid appears pink and is localized to the perifollicular zone. (B) The identical area exhibits green birefringence in polarized light. Amyloid is indicated by arrows.</p

Changes in natural killer and T lymphocyte phenotypes in response to cardiovascular risk management

The pro-inflammatory and regulatory roles of T lymphocytes in atherosclerosis are well established but less is known about natural killer (NK) cells and natural killer T (NKT)-like cells. The effects of cardiovascular risk management on the phenotypes of these cells are unknown. To assess changes in NK cell and lymphocyte phenotypes and circulating inflammatory proteins in response to cardiovascular risk management in patients with carotid atherosclerosis. Fifty patients were included in a prospective clinical study. Measurements were at baseline and after 12 months of cardiovascular risk management. Circulating NK, NKT-like and T lymphocyte subpopulations were phenotyped by multi-colour flow cytometry. Proximity extension assay was performed for 176 plasma proteins associated with inflammation and cardiovascular disease. At 12 months there were significant reductions in LDL (P=0.001) and blood pressure (P=0.028). NK cells responded with a reduction in pro-inflammatory (NKG2C(+)) cells (P=0.0003), an increase in anti-inflammatory (NKG2A(+)) cells (P=0.032), and a reduction in terminally differentiated (CD57(+)) NK cells. NKT-like cells showed a similar decrease in terminally differentiated subpopulations (P=0.000002). Subpopulations of T helper cells exhibited a significant reduction in central memory (P=1.09x10(-8)) and a significant increase in CD4(+) naive- (P=0.0008) and effector memory T cells (P=0.006). The protein analysis indicated that cardiovascular risk management affects proteins involved in the inflammatory NF-kappa B pathway. The consistent decrease in senescent phenotypes of NK, NKT-like and CD4(+) cells with a concomitant increase in more naive, phenotypes suggests a change towards a less pro-inflammatory lymphocyte profile in response to cardiovascular risk management.Trial registry name: CARotid MRI of Atherosclerosis (CARMA). ClinicalTrials.gov identifier NCT04835571 (08/04/2021). https://www.clinicaltrials.gov/study/NCT04835571.Funding Agencies|Linkoping University; Region Ostergotland [RO-610581]; Henry och Ella Margareta Stahls Stiftelse (Henry and Ella Margareta Stahl's Foundation) [LIO-748491]; Forskningsradet i Sydostra Sverige [FORSS-756191]</p

Major alterations to monocyte and dendritic cell subsets lasting more than 6 months after hospitalization for COVID-19

Introduction: After more than two years the Coronavirus disease-19 (COVID-19) pandemic continues to burden healthcare systems and economies worldwide, and it is evident that the effects on the immune system can persist for months post-infection. The activity of myeloid cells such as monocytes and dendritic cells (DC) is essential for correct mobilization of the innate and adaptive responses to a pathogen. Impaired levels and responses of monocytes and DC to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is likely to be a driving force behind the immune dysregulation that characterizes severe COVID-19. Methods: Here, we followed a cohort of COVID-19 patients hospitalized during the early waves of the pandemic for 6-7 months. The levels and phenotypes of circulating monocyte and DC subsets were assessed to determine both the early and long-term effects of the SARS-CoV-2 infection. Results: We found increased monocyte levels that persisted for 6-7 months, mostly attributed to elevated levels of classical monocytes. Myeloid derived suppressor cells were also elevated over this period. While most DC subsets recovered from an initial decrease, we found elevated levels of cDC2/cDC3 at the 6-7 month timepoint. Analysis of functional markers on monocytes and DC revealed sustained reduction in program death ligand 1 (PD-L1) expression but increased CD86 expression across almost all cell types examined. Finally, C-reactive protein (CRP) correlated positively to the levels of intermediate monocytes and negatively to the recovery of DC subsets. Conclusion: By exploring the myeloid compartments, we show here that alterations in the immune landscape remain more than 6 months after severe COVID-19, which could be indicative of ongoing healing and/or persistence of viral antigens.Funding: ML SciLifeLab/KAW COVID-19 Research Program, Swedish Research Council [201701091]; COVID-19 ALF (Linkoeping University Hospital Research Fund), Region OEstergoetland ALF Grant [ROE935411]; Regional ALF Grant; Vrinnevi Hospital in Norrkoeping</p