Весовые соотношения тимуса и надпочечников у антенатально погибших плодов

ТИМУСНАДПОЧЕЧНИКИтимомегалиядети первого года жизнидети раннего возрастаМЛАДЕНЕЦ, СМЕРТНОСТЬЭНДОКРИННОЙ СИСТЕМЫ БОЛЕЗНИСМЕРТНОСТЬ ДЕТСКАЯсиндром внезапной детской смерт

SHIP deficiency is associated with the Crohn’s disease susceptibility variant in ATG16L1 and leads to increased interleukin-1beta transcription and intestinal autoinflammation

Crohn`s disease (CD) is a polygenic immune-mediated disease of the gastrointestinal tract characterized by chronic inflammation. The SH2-domain-containing inositol polyphosphate 5΄phosphatase (SHIP) is a hematopoietic-specific negative regulator of inflammatory cytokine production and plays an important role in regulating immune homeostasis. Using the SHIP deficient mouse model of intestinal inflammation, we found that IL-1β is increased in SHIP-/- mouse macrophages due to increased class I PI3K p110α activity. Macrophage depletion or treatment with an IL-1 receptor antagonist reduced development of intestinal inflammation in SHIP-/- mice. To interrogate if SHIP is dysregulated in people with ileal CD, we demonstrate that subjects with ileal CD have reduced SHIP mRNA expression and enzymatic activity at sites of inflammation and in PBMCs, compared to control subjects. A single nucleotide polymorphism (SNP) in the gene encoding ATG16L1 (T300A) causes an autophagy defect and is associated with increased IL-1β production and susceptibility to CD. In all tissues from our patient cohort and in PBMCs from a second healthy control cohort, subjects, who were homozygous for the CD-associated ATG16L1 T300A encoding gene variant, had reduced SHIP mRNA expression and enzymatic activity, which correlated with increased IL-1β production. In addition, starvation-induced autophagy increased SHIP protein levels, which were reduced in the presence of the ATG16L1 CD-associated risk allele. Examining the effects of additional autophagy and CD-related gene variants, which may affect SHIP mRNA expression and enzymatic activity, on IL-1β production in PBMCs from a cohort of healthy control subjects, we found that the NOD2 rs2066844 and the XBP-1 rs35873774 gene variants were associated with increased IL-1β production in response to specific PAMPs. Collectively, these data identify SHIP up-regulation as a novel mechanism by which autophagy regulates IL-1β production and intestinal autoinflammation. Our findings also identify a subgroup of CD patients that could be amenable to treatment with therapy that targets IL-1β.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat

Apoptotic neutrophils augment the inflammatory response to Mycobacterium tuberculosis infection in human macrophages

Macrophages in the lung are the primary cells being infected by Mycobacterium tuberculosis (Mtb) during tuberculosis. Innate immune cells such as macrophages and neutrophils are first recruited to the site of infection, and mount the early immune protection against this intracellular pathogen. Neutrophils are short-lived cells and removal of apoptotic cells by resident macrophages is a key event in the resolution of inflammation and tissue repair. Such anti-inflammatory activity is not compatible with effective immunity to intracellular pathogens. We therefore investigated how uptake of apoptotic neutrophils by Mtb-activated human monocyte-derived macrophages modulates their function. We show that Mtb infection exerts a potent pro-inflammatory activation of human macrophages with enhanced gene activation and release of several cytokines (TNF, IL-1ß, IL-6, IL-18 and IL-10). This response was augmented by apoptotic neutrophils. Macrophages containing both Mtb and apoptotic cells showed a stronger cytokine expression than non-infected cells. The enhanced macrophage response is linked to apoptotic neutrophil-driven activation of the NLRP3 inflammasome and subsequent IL-1β signalling. We also demonstrate that apoptotic neutrophils not only modulate the inflammatory response, but also enhance the capacity of infected macrophages to control intracellular growth of virulent Mtb. Taken together, these results suggest a novel role for apoptotic neutrophils in the modulation of the macrophage-dependent inflammatory response, which can contribute to the early control of Mtb infection

Uptake of Mtb and PMNapo.

<p>hMDMs were stimulated with FITC labeled γ-irr Mtb with or without subsequent stimulation with PKH26-labeled PMNapo. The uptake of Mtb and PMNapo was analyzed by flow cytometry. Values represent percentage of positive cells ± SEM (n = 5).</p

The augmentation of hMDM activation is dependent specifically on phagocytosis of apoptotic neutrophils.

<p>hMDMs were pre-incubated with or without cytochalasin D (CytD) prior to stimulation with γ-irr Mtb at a ratio of 5∶1 alone (Mtb) or together with PMNapo at a ratio of 2∶1 (Mtb + PMNapo) for one hour where after non-ingested prey were removed and the hMDMs were cultured for 18 h. Data are expressed as mean TNFα released <u>+</u> SEM (n = 3).</p

Apoptotic neutrophils augment caspase-1 activation in Mtb-stimulated hMDMs.

<p>hMDMs were stimulated with γ-irr Mtb for one hour with or without subsequent stimulation with PMNapo or Jurkatapo at a ratio of 2∶1 for one hour where after non-ingested prey were removed and the hMDMs were cultured for 3 h. hMDMs were extensively washed and lysed in Laemmli sample buffer. (A) Representative SDS-PAGE immunoblots of hMDM cell lysates probed with anti-caspase-1 antibodies, detecting both unprocessed inactive caspase-1 (p45) and cleaved activated caspase-1 (p20). Membranes were then stripped and re-probed for β-actin as a control for equal loading. (B) Caspase-1 activity shown as the ratio between caspase-1 p20/caspase-1 p45 and data expressed as mean + SEM (n = 4-5). Differences between groups were calculated using paired sample Student t-test, and significant differences are shown as * (p<0.05); ns, not significant.</p

Uptake of apoptotic neutrophils increases the number of IL-1β producing cells in the infected cell population.

<p>hMDMs were stimulated with FITC labeled γ-irr Mtb at a ratio of 5∶1 (Mtb), with subsequent stimulation with PKH26-labeled PMNapo at a ratio of 2∶1 (Mtb+PMNapo). The hMDMs were stained for intracellular IL-1β (Pacific Blue) and TNFα (Alexa Fluor 647) and analyzed by flow cytometry. (A) The figure shows shows the percentage of hMDMs (based on forward/side scatter) positive for Mtb (FITC/FL-1), PMNapo (PKH26/FL-2) or both. (B) The histograms show the percentage of TNFα (Alexa 647/FL-6) or IL-1β (Pacific Blue/FL-9) positive cells in the population of hMDMs which have phagocytosed both Mtb and PMNapo. (C) The figure shows the number of IL-1β or TNFα producing cells (%) in the following subpopulations; FITC⁻/PKH26⁻ (no uptake), FITC⁻/PKH26⁺ (PMNapo uptake), FITC⁺/PKH26⁻ (Mtb uptake) and FITC⁺/PKH26⁺ (Mtb+PMNapo). (n = 5). Differences between groups are shown as * (p<0.05).</p

Apoptotic neutrophils enhance the hMDM capacity to control intracellular Mtb.

<p>hMDMs were infected with Mtb H37Rv and uptake was determined (D0). Following infection, hMDMs were stimulated with PMNapo or Jurkatapo at a ratio of 2∶1. Measurements of Mtb by luminometry were performed at indicated time points. Data are presented as ratio compared to the initial bacterial load at D0 (<i>i.e.</i> increase in bacterial load) and the graphs show the mean <u>+</u> SEM (n = 5). Differences between groups are shown as * (p<0.05), ** (p<0.01) or *** (p<0.001).</p

Apoptotic neutrophils augmentation of cytokine release depends on caspase-1 activity.

<p>hMDMs, with or without pre-incubation with Ac-YVAD-CMK (caspase-1 inhibitor), were stimulated with γ-irr Mtb with or without PMNapo at a ratio of 2∶1 for one hour where after non-ingested prey were removed and the hMDMs were cultured for 18 h. Cytokine levels were measured by cytometric bead array, and data are expressed as mean cytokine released <u>+</u> SEM (n = 6). Differences between groups are shown as * (p<0.05), ** (p<0.01) or *** (p<0.001).</p

Apoptotic neutrophils augment the inflammatory response of macrophages to Mtb.

<p>hMDMs were stimulated with γ-irr Mtb at a ratio of 5∶1 (Mtb) for one hour, with or without subsequent stimulation with PMNapo at a ratio of 2∶1 (Mtb+PMNapo) for one hour, where after the hMDMs were re-cultured for 20 h. hMDMs stimulated with PMNapo without prior stimulation with Mtb (PMNapo) was included as control. mRNA levels were measured by quantitative real-time PCR. Data are presented as ratios over untreated cells (NC) <u>+</u> SEM (n = 7). Differences between groups are shown as * (p<0.05), ** (p<0.01) or *** (p<0.001).</p