Matrix metalloproteinases (MMPs) are transcriptionally regulated proteases that have multiple roles in modifying the extracellular matrix (ECM) and inflammatory response. Our previous work identified Mmp28 as a key regulator of inflammation and macrophage polarization during experimental models of pulmonary infection, fibrosis, and chronic smoke exposure. However, the signaling pathways responsible for regulation of macrophage Mmp28 expression remain undefined. This study utilized murine macrophages obtained from wild-type, Tlr2−/−, Tlr4−/−, MyD88−/−, Ticam1Lps2 (Trifmutant), and Ifnar1−/− mice to test the hypothesis that macrophage Mmp28 expression was dependent on TRIF and type I IFN. Our results support the hypothesis, demonstrating that increased macrophage Mmp28 expression was dependent on type I IFN following LPS and poly(I:C) stimulation. To gain further insight into the function of MMP28, we explored the inflammatory response of macrophages derived from wild-type or Mmp28−/−mice to stimulation with poly(I:C). Our data support a role for MMP28 in regulating the macrophage inflammatory response to poly(I:C), as expression of Ccl2, Ccl4, Cxcl10, and Il6 were increased in Mmp28−/− macrophages. Together, these data support a model in which macrophages integrate TRIF- and type I IFN-dependent signaling to coordinate regulation of proteins with the capacity to modify the ECM.

Background Mer receptor tyrosine kinase (MerTK) is key for efficient phagocytosis of apoptotic neutrophils (ANs) and homeostasis of IL-10 production by human anti-inflammatory M2c monocytes/macrophages. We asked whether stimulation of certain M2c surface receptors contribute in turn to MerTK activation. Methods Human monocytes/macrophages were differentiated under M1, M2a and M2c polarizing conditions. We tested the effects of antibody-mediated cross-linking of M2c receptors (i.e., CD14, CD16, CD32, CD163, CD204) on MerTK phosphorylation and phagocytosis of ANs. MerTK expression was also studied by flow cytometry and western blot in the presence of LPS and in M2c-derived microvesicles (MVs). Results Antibody cross-linking of either CD14 or CD32/FcγRII led to Syk activation and MerTK phosphorylation in its two distinct glycoforms (175–205 and 135–155 KDa). Cross-linked CD14 enhanced efferocytosis by M2c macrophages and enabled M1 and M2a cells to clear ANs efficiently. In M1 conditions, LPS abolished surface MerTK expression on CD14bright cell subsets, so disrupting the anti-inflammatory pathway. In M2c cells, instead, MerTK was diffusely and brightly co-expressed with CD14, and was also detected in M2c macrophage-derived MVs; in these conditions, LPS only partially down-regulated MerTK on the cell surface, while the smaller MerTK glycoform contained in MVs remained intact. Conclusions Cooperation between CD14 and MerTK fosters tethering and engulfment of ANs by human monocytes/macrophages. CD14 stands between M1-related LPS co-receptor activity and M2c-related MerTK-dependent responses. MerTK interaction with CD32/FcγRII, its detection in M2c MVs, and the differential localization and LPS susceptibility of MerTK glycoforms add further new elements to the complexity of the MerTK network. Acknowledgements NIAID and Lupus Research Alliance

Background Two major therapies for cutaneous lupus erythematosus (CLE) are the antimalarials, hydroxychloroquine (HCQ) and quinacrine (QC). HCQ is often the first-line therapy for CLE, but only half of patients show a response to it. While some of the patients that do not initially respond to HCQ benefit from the addition of QC, there is a subset of patients that are refractory to both antimalarials. Refractoriness poses a huge challenge because these patients will often continue to have active disease when they are initially started on antimalarials. To better characterize these refractory patients, we investigated the immunologic characteristics of patients that respond to antimalarials versus those that do not. Methods CLE patients were classified as HCQ-responders, HCQ/QC-responders, or HCQ/QC-nonresponders. Immunohistochemistry was used to characterize the inflammatory cell composition and cytokine expression in lesional skin biopsies from patients. Total RNA was extracted from these biopsies to analyze specific gene signatures. The patient’s CLASI score – a measure of disease activity – at the time of the biopsy was also determined. Results Immunohistochemistry showed that myeloid dendritic cells (mDCs) were significantly higher in HCQ/QC-responders compared to HCQ-responders and HCQ/QC-nonresponders, while plasmacytoid dendritic cells, neutrophils, macrophages, and autoreactive T cells did not differ significantly among the three groups. The HCQ/QC-nonresponder group was distinct from the other groups in that their CLASI scores did correlate positively with the number of macrophages (p Conclusions An increased number of mDCs may contribute to HCQ-refractoriness and predict a better response to treatment with both HCQ and QC but do not contribute to HCQ/QC-refractoriness. The significant correlation between macrophages and CLASI scores in the HCQ/QC-nonresponders, a finding not seen in either HCQ or HCQ/QC-responders, may also indicate that macrophages are more involved in antimalarial-refractory skin disease. The difference between the responders and nonresponders is further confirmed by the cytokine staining and mRNA expression. Our data is an initial step in determining the activation pathways that account for the lack of response to antimalarials. Acknowledgements This work was supported by National Institutes of Health [K24 AR002207] and Veterans Affairs Merit Review [I01B × 000706] to Dr. Victoria Werth.

Lupus nephritis (LN) is a multifactorial event that contributes to the long-term mortality of systemic lupus erythematosus (SLE). Activation of NLRP3 inflammasome has been known to play a role in SLE pathogenesis. We evaluated the renal protection effects of procyanidin B2 (PCB2) and the involvement of NLRP3 in a mouse model involving MRL/lpr and MRL/MpJ mice. Kidney injury was evaluated by measuring the renal clinical and pathological features, renal immune complex deposition, and serum anti-double-stranded (anti-dsDNA) Abs. ELISA and Western blotting were used to detect NLRP3 inflammasome activation and IL-1β/IL-18 production. NLRP3 gene silencing was introduced into MRL/lpr mice by short hairpin RNA, and the renal damage was compared with the treatment of PCB2. PCB2 remarkably reduced renal damage in MRL/lpr mice, reflected by the reduced proteinuria, and serum levels of blood urea nitrogen and creatinine, as well as pathological features with less renal injury. PCB2 significantly reduced renal immune complex deposition and serum anti-dsDNA levels, notably inhibited the NLRP3 inflammasome activation, and reduced the renal and serum levels of IL-1β and IL-18 in MRL/lpr mice compared with those of NLRP3 gene-silenced MRL-lpr mice. PCB2 significantly suppressed LN in MRL-lpr mice by inhibiting the activation of NLRP3 inflammasome and subsequent IL-1β and IL-18 production. This finding explores a novel mechanism by which procyanidin exerts inflammatory suppression effects and its clinical benefits in LN prevention.

Innate immunity natural Abs (NAbs) execute a number of functions, including protection and surveillance. Despite active research, the stimuli that induce the formation of NAbs are still described only hypothetically. Here, we compared repertoires of anti-glycan Abs in the peripheral blood of mice that received per os various bacteria. The repertoires of Abs of mice primed in this way were compared using a microarray that included about 350 glycans, as well as 150 bacterial polysaccharides. Sterile mice did not possess anti-glycan Abs. Oral inoculation of a single strain or combination of two to four strains of bacteria, as well as putting the animals on short-term nutrition with non-sterile food, did not contribute significantly to the formation of Abs, whereas a single gavage of digested food of non-sterile mice induced the formation of a repertoire close to the natural ones. Interestingly, the priming with polysaccharide Ags (in a composition of the bacterial cell envelope), that is, dominant Ags of bacteria, led to the induction of Abs against typical glycans of mammalian glycoproteins and glycolipids (e.g. Abs of the ABH blood group system) that do not have a structural similarity to the polysaccharides. The results support the importance of early contact with a naïve immune system with microorganisms of the environment to form a normal NAbs repertoire.

The aim of this study is to describe the prevalence of single single-nucleotide polymorphisms (SNPs) as well as their combinations in genes encoding proteins involved in the immune response in children with bacterial meningitis. The prospective study group consisted of 39 children with bacterial meningitis and 49 family members surveyed between 2012 and 2016. Eleven SNPs in seven genes involved in immune response were analysed. The mean number of minor frequency alleles (MAF) of studied SNPs was lowest in the control group and highest in patients with pneumococcal meningitis. We found that carrying ≥6 MAF of studied SNPs was associated with an increased risk of pneumococcal meningitis. The prevalence of risky variants was noted to be higher in patients with pneumococcal meningitis as compared to the control group. In conclusion, genetic factors are a relevant factor in determining the susceptibility to bacterial meningitis. A statistically significant cumulative effect of mutated variants on increasing the risk of bacterial meningitis was detected. Combining all three SNPs in MBL2 improves the prediction of susceptibility to pneumococcal meningitis. Analysis of risky alleles can help indicate people prone to the disease who are ‘gene-immunocompromised’.

Viral infection is a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplant (HSCT), with up to one in four deaths directly linked to viral disease. Whilst awaiting lymphocyte reconstitution post-HSCT, the innate antiviral immune response is the first line of defense against invading viruses. Several novel innate viral-sensing pathways have recently been characterized, but their physiological importance in humans is poorly understood. We analyzed a panel of innate viral-sensor genes in HSCT patients, and assessed whether differences in innate antiviral responses could account for variation in susceptibility to viral infections. Expression levels of innate viral sensors in HSCT patients with active viral infections, HSCT patients without active infections and healthy volunteers were highly homogenous. Although IFN-α expression was up-regulated in actively infected patients relative to controls, a corresponding up-regulation of innate viral sensor expression was not observed. IFN-α stimulation of patient PBMCs in vitro showed intact IFN-α signaling, but actively infected patients' PBMCs had reduced up-regulation of innate viral sensors. We show that the aberrant IFN-α responses in HSCT patients were not due to calcineurin inhibition. Our data therefore raises the possibility of an intrinsic defect in innate viral sensor up-regulation in HSCT patients following viral infection.

Automobile traffic, industrial processes and natural phenomena cause notable air pollution, including gaseous and particulate contaminants, in urban centers. Exposure to particulate matter (PM) air pollution affects human health, and has been linked to respiratory, cardiovascular and neurological diseases. The mechanisms underlying inflammation in these diverse diseases, and to what extent health effects are different for PM obtained from different sources or locations, are still unclear. This study investigated the invitro toxicity of ambient course (PM10) and fine (PM2.5) particulate matter collected at seven sites in the urban and periurban zones of Quito, Ecuador. Material from all sites was capable of activating TLR2 and TLR4 signaling pathways, with differences in the activation related to particle size. Additionally, airborne particulate matter from Quito is an effective activator of the NLRP3 inflammasome.

The pro-inflammatory potency and causal relationship with asthma of inhaled endotoxins have underscored the importance of accurately assessing the endotoxin content of organic dusts. The Limulus amebocyte lysate (LAL) assay has emerged as the preferred assay, but its ability to measure endotoxin in intact bacteria and organic dusts with similar sensitivity as purified endotoxin is unknown. We used metabolically radiolabeled Neisseria meningitidis and both rough and smooth Escherichia coli to compare dose-dependent activation in the LAL with purified endotoxin from these bacteria and shed outer membrane (OM) blebs. Labeled [14C]-3-OH-fatty acids were used to quantify the endotoxin content of the samples. Purified meningococcal and E. coli endotoxins and OM blebs displayed similar specific activity in the LAL assay to the purified LPS standard. In contrast, intact bacteria exhibited fivefold lower specific activity in the LAL assay but showed similar MD-2-dependent potency as purified endotoxin in inducing acute airway inflammation in mice. Pre-treatment of intact bacteria and organic dusts with 0.1 M Tris-HCl/10 mM EDTA increased by fivefold the release of endotoxin. These findings demonstrate that house dust and other organic dusts should be extracted with Tris/EDTA to more accurately assess the endotoxin content and pro-inflammatory potential of these environmental samples.

BMAL1 (brain and muscle ARNT-like protein 1, also known as MOP3 or ARNT3) belongs to the family of the basic helix-loop-helix (bHLH)-PAS domain-containing transcription factors, and is a key component of the molecular oscillator that generates circadian rhythms. Here, we report that BMAL1-deficient cells are significantly more susceptible to infection by two major respiratory viruses of the Paramyxoviridae family, namely RSV and PIV3. Embryonic fibroblasts from Bmal1-/- mice produced nearly 10-fold more progeny virus than their wild type controls. These results were supported by animal studies whereby pulmonary infection of RSV produced a more severe disease and morbidity in Bmal1-/-mice. These results show that BMAL1 can regulate cellular innate immunity against specific RNA viruses.