Intestinal microbiome and metabolome signatures in patients with chronic granulomatous disease.

BACKGROUND: Chronic granulomatous disease (CGD) is caused by defects in any 1 of the 6 subunits forming the nicotinamide adenine dinucleotide phosphate oxidase complex 2 (NOX2), leading to severely reduced or absent phagocyte-derived reactive oxygen species production. Almost 50% of patients with CGD have inflammatory bowel disease (CGD-IBD). While conventional IBD therapies can treat CGD-IBD, their benefits must be weighed against the risk of infection. Understanding the impact of NOX2 defects on the intestinal microbiota may lead to the identification of novel CGD-IBD treatments. OBJECTIVE: We sought to identify microbiome and metabolome signatures that can distinguish individuals with CGD and CGD-IBD. METHODS: We conducted a cross-sectional observational study of 79 patients with CGD, 8 pathogenic variant carriers, and 19 healthy controls followed at the National Institutes of Health Clinical Center. We profiled the intestinal microbiome (amplicon sequencing) and stool metabolome, and validated our findings in a second cohort of 36 patients with CGD recruited through the Primary Immune Deficiency Treatment Consortium. RESULTS: We identified distinct intestinal microbiome and metabolome profiles in patients with CGD compared to healthy individuals. We observed enrichment for Erysipelatoclostridium spp, Sellimonas spp, and Lachnoclostridium spp in CGD stool samples. Despite differences in bacterial alpha and beta diversity between the 2 cohorts, several taxa correlated significantly between both cohorts. We further demonstrated that patients with CGD-IBD have a distinct microbiome and metabolome profile compared to patients without CGD-IBD. CONCLUSION: Intestinal microbiome and metabolome signatures distinguished patients with CGD and CGD-IBD, and identified potential biomarkers and therapeutic targets.

Evolution of Mycobacterium abscessus in the human lung: Cumulative mutations and genomic rearrangement of porin genes in patient isolates.

BACKGROUND: Mycobacterium abscessus subspecies massiliense (M. massiliense) is increasingly recognized as an emerging bacterial pathogen, particularly in cystic fibrosis (CF) patients and CF centres' respiratory outbreaks. We characterized genomic and phenotypic changes in 15 serial isolates from two CF patients (1S and 2B) with chronic pulmonary M. massiliense infection leading to death, as well as four isolates from a CF centre outbreak in which patient 2B was the index case. RESULTS: Comparative genomic analysis revealed the mutations affecting growth rate, metabolism, transport, lipids (loss of glycopeptidolipids), antibiotic susceptibility (macrolides and aminoglycosides resistance), and virulence factors. Mutations in 23S rRNA, mmpL4, porin locus and tetR genes occurred in isolates from both CF patients. Interestingly, we identified two different spontaneous mutation events at the mycobacterial porin locus: a fusion of two tandem porin paralogs in patient 1S and a partial deletion of the first porin paralog in patient 2B. These genomic changes correlated with reduced porin protein expression, diminished 14C-glucose uptake, slower bacterial growth rates, and enhanced TNF-α induction in mycobacteria-infected THP-1 human cells. Porin gene complementation of porin mutants partly restored 14C-glucose uptake, growth rate and TNF-α levels to those of intact porin strains. CONCLUSIONS: We hypothesize that specific mutations accumulated and maintained over time in M. massiliense, including mutations shared among transmissible strains, collectively lead to more virulent, host adapted lineages in CF patients and other susceptible hosts.

Hepatocyte cannabinoid 1 receptor nullification alleviates toxin-induced liver damage via NF-κB signaling.

Cannabinoid 1 receptor (CB1R) expression is upregulated in the liver with viral hepatitis, cirrhosis, and both alcoholic and non-alcoholic fatty liver disease (FLD), whereas its expression is muted under usual physiological conditions. Inhibiting CB1R has been shown to be beneficial in preserving hepatic function in FLD but it is unclear if inhibiting CB1R during an inflammatory response to an acute hepatic injury, such as toxin-induced injury, would also be beneficial. We found that intrinsic CB1R in hepatocytes regulated liver inflammation-related gene transcription. We tested if nullification of hepatocyte-specific CB1R (hCNR1-/-) in mice protects against concanavalin A (Con A)-induced liver injury. We looked for evidence of liver damage and markers of inflammation in response to Con A by measuring liver enzyme levels and proinflammatory cytokines (e.g., TNF-α, IL-1ß, IL-6, IL-17) in serum collected from hCNR1-/- and control mice. We observed a shift to the right in the dose-response curve for liver injury and inflammation in hCNR1-/- mice. We also found less inflammatory cell infiltration and focal necrosis in livers of hCNR1-/- mice compared to controls, resulting from downregulated apoptotic markers. This anti-apoptotic mechanism results from increased activation of nuclear factor kappa B (NF-κB), especially cAMP-dependent cannabinoid signaling and membrane-bound TNF-α, via downregulated TNF-α receptor 2 (TNFR2) transcription levels. Collectively, these findings provide insight into involvement of CB1R in the pathogenesis of acute liver injury.

Drug-drug Interactions in Patients with HIV and Cancer in Sub-Saharan Africa.

In Sub-Saharan Africa, the cancer burden is predicted to increase by > 85% by 2030, the largest increase worldwide. This region has a large HIV-positive population. Drug-drug interactions (DDIs) from concomitant use of multiple drugs increase the risk of drug toxicities, sub-optimal therapy, and drug resistance. With the increase in polypharmacy, involving antiretroviral (ARV), and anticancer drugs, there is a greater need for an appreciation of clinically relevant DDIs. Anticancer and ARV drugs studied in this review were from The World Health Organization's Model List of Essential Medicines 2017. We reviewed; drug package inserts, www.drugbank.ca and www.UpToDate.com, to evaluate pharmacokinetic interactions with cytochrome P450 (CYP450) and ABCB1. The DDIs between drugs were assessed using the University Of Liverpool, UK HIV Drug Interactions Checker, and the LexiComp Drug Interaction tool of www.UpToDate.com. About 70% of ARVs studied interact with CYP450, all involve CYP3A4, and 55% interact with ABCB1. About 65% of anticancer drugs interact with CYP450, 44% of which do so through CYP3A4. About 75% of anticancer drugs interact with ARV drugs, with nine absolute contraindications to concomitant therapy. There exist a substantial number of DDIs between ARV and anticancer drugs, primarily mediated through CYP450 enzymes. Dolutegravir based regimens offer the safest DDI profile for concurrent use with anticancer drugs. However, there are substantial gaps in our knowledge, and this study serves to highlight the need for additional research to better define these interactions and their effect on drug exposure, as attention to these DDIs is a relatively simple intervention that could lead to optimizing disease treatment.

Immune Complex-Driven Generation of Human Macrophages with Anti-Inflammatory and Growth-Promoting Activity.

To maintain homeostasis, macrophages must be capable of assuming either an inflammatory or an anti-inflammatory phenotype. To better understand the latter, we stimulated human macrophages in vitro with TLR ligands in the presence of high-density immune complexes (IC). This combination of stimuli resulted in a broad suppression of inflammatory mediators and an upregulation of molecules involved in tissue remodeling and angiogenesis. Transcriptomic analysis of TLR stimulation in the presence of IC predicted the downstream activation of AKT and the inhibition of GSK3. Consequently, we pretreated LPS-stimulated human macrophages with small molecule inhibitors of GSK3 to partially phenocopy the regulatory effects of stimulation in the presence of IC. The upregulation of DC-STAMP and matrix metalloproteases was observed on these cells and may represent potential biomarkers for this regulatory activation state. To demonstrate the presence of these anti-inflammatory, growth-promoting macrophages in a human infectious disease, biopsies from patients with leprosy (Hanseniasis) were analyzed. The lepromatous form of this disease is characterized by hypergammaglobulinemia and defective cell-mediated immunity. Lesions in lepromatous leprosy contained macrophages with a regulatory phenotype expressing higher levels of DC-STAMP and lower levels of IL-12, relative to macrophages in tuberculoid leprosy lesions. Therefore, we propose that increased signaling by FcγR cross-linking on TLR-stimulated macrophages can paradoxically promote the resolution of inflammation and initiate processes critical to tissue growth and repair. It can also contribute to infectious disease progression.

Regulatory Macrophages Inhibit Alternative Macrophage Activation and Attenuate Pathology Associated with Fibrosis.

Diversity and plasticity are the hallmarks of macrophages. The two most well-defined macrophage subsets are the classically activated macrophages (CAMϕs) and the IL-4-derived alternatively activated macrophages (AAMϕs). Through a series of studies, we previously identified and characterized a distinct population of macrophages with immunoregulatory functions, collectively termed regulatory macrophages (RMϕs). Although considerable advances have been made in understanding these various macrophage subsets, it is not known whether macrophages of one activation state can influence the other. In this study, we examined whether RMϕs capable of inhibiting inflammatory responses of CAMϕs could also inhibit AAMϕs and their profibrotic responses. Our results demonstrated that RMϕs significantly dampened the alternate activation phenotype of AAMϕs generated in vitro and intrinsically occurring AAMϕs from TACI-/- macrophages. Further, RMϕs inhibited AAMϕ-promoted arginase activity and fibroblast proliferation in vitro. This inhibition occurred regardless of the strength, duration, and mode of alternative activation and was only partially dependent on IL-10. In the chlorhexidine gluconate-induced peritoneal fibrosis model, AAMϕs worsened the fibrosis, but RMϕs rescued mice from AAMϕ-mediated pathological conditions. Taken together, our study demonstrates that RMϕs are a specialized subset of macrophages with a nonredundant role in limiting overt proregenerative functions of AAMϕs, a role distinct from their well-defined role of suppression of inflammatory responses by CAMϕs.

Macrophages and the Recovery from Acute and Chronic Inflammation.

In recent years, researchers have devoted much attention to the diverse roles of macrophages and their contributions to tissue development, wound healing, and angiogenesis. What should not be lost in the discussions regarding the diverse biology of these cells is that when perturbed, macrophages are the primary contributors to potentially pathological inflammatory processes. Macrophages stand poised to rapidly produce large amounts of inflammatory cytokines in response to danger signals. The production of these cytokines can initiate a cascade of inflammatory mediator release that can lead to wholesale tissue destruction. The destructive inflammatory capability of macrophages is amplified by exposure to exogenous interferon-γ, which prolongs and heightens inflammatory responses. In simple terms, macrophages can thus be viewed as incendiary devices with hair triggers waiting to detonate. We have begun to ask questions about how these cells can be regulated to mitigate the collateral destruction associated with macrophage activation.

Complement-mediated 'bystander' damage initiates host NLRP3 inflammasome activation.

Complement activation has long been associated with inflammation, primarily due to the elaboration of the complement anaphylotoxins C5a and C3a. In this work, we demonstrate that the phagocytosis of complement-opsonized particles promotes host inflammatory responses by a new mechanism that depends on the terminal complement components (C5b-C9). We demonstrate that during the phagocytosis of complement-opsonized particles, the membrane attack complex (MAC) of complement can be transferred from the activating particle to the macrophage plasma membrane by a 'bystander' mechanism. This MAC-mediated bystander damage initiates NLRP3 inflammasome activation, resulting in caspase-1 activation and IL-1ß and IL-18 secretion. Inflammasome activation is not induced when macrophages phagocytize unopsonized particles or particles opsonized with serum deficient in one of the terminal complement components. The secretion of IL-1ß and IL-18 by macrophages depends on NLRP3, ASC (also known as PYCARD) and caspase-1, as macrophages deficient in any one of these components fail to secrete these cytokines following phagocytosis. The phagocytosis of complement-opsonized particles increases leukocyte recruitment and promotes T helper 17 cell (TH17) biasing. These findings reveal a new mechanism by which complement promotes inflammation and regulates innate and adaptive immunity.

The generation of macrophages with anti-inflammatory activity in the absence of STAT6 signaling.

Macrophages readily change their phenotype in response to exogenous stimuli. In this work, macrophages were stimulated under a variety of experimental conditions, and phenotypic alterations were correlated with changes in gene expression. We identified 3 transcriptionally related populations of macrophages with immunoregulatory activity. They were generated by stimulating cells with TLR ligands in the presence of 3 different "reprogramming" signals: high-density ICs, PGE2, or Ado. All 3 of these cell populations produced high levels of transcripts for IL-10 and growth and angiogenic factors. They also secreted reduced levels of inflammatory cytokines IL-1ß, IL-6, and IL-12. All 3 macrophage phenotypes could partially rescue mice from lethal endotoxemia, and therefore, we consider each to have anti-inflammatory activity. This ability to regulate innate-immune responses occurred equally well in macrophages from STAT6-deficient mice. The lack of STAT6 did not affect the ability of macrophages to change cytokine production reciprocally or to rescue mice from lethal endotoxemia. Furthermore, treatment of macrophages with IL-4 failed to induce similar phenotypic or transcriptional alterations. This work demonstrates that there are multiple ways to generate macrophages with immunoregulatory activity. These anti-inflammatory macrophages are transcriptionally and functionally related to each other and are quite distinct from macrophages treated with IL-4.

Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.

BACKGROUND: Mutations in signal transducer and activator of transcription (STAT) 1 cause a broad spectrum of disease, ranging from severe viral and bacterial infections (amorphic alleles) to mild disseminated mycobacterial disease (hypomorphic alleles) to chronic mucocutaneous candidiasis (CMC; hypermorphic alleles). The hypermorphic mutations are also associated with arterial aneurysms, autoimmunity, and squamous cell cancers. OBJECTIVE: We sought to investigate the role of STAT1 gain-of-function mutations in phenotypes other than CMC. METHODS: We initially screened patients with CMC and autoimmunity for STAT1 mutations. We functionally characterized mutations in vitro and studied immune profiles and regulatory T (Treg) cells. After our initial case identifications, we explored 2 large cohorts of patients with wild-type forkhead box protein 3 and an immune dysregulation-polyendocrinopathy-enteropathy-X-linked (IPEX)-like phenotype for STAT1 mutations. RESULTS: We identified 5 children with polyendocrinopathy, enteropathy, and dermatitis reminiscent of IPEX syndrome; all but 1 had a variety of mucosal and disseminated fungal infections. All patients lacked forkhead box protein 3 mutations but had uniallelic STAT1 mutations (c.629 G>T, p.R210I; c.1073 T>G, p.L358W, c.796G>A; p.V266I; c.1154C>T, T385M [2 patients]). STAT1 phosphorylation in response to IFN-γ, IL-6, and IL-21 was increased and prolonged. CD4(+) IL-17-producing T-cell numbers were diminished. All patients had normal Treg cell percentages in the CD4(+) T-cell compartment, and their function was intact in the 2 patients tested. Patients with cells available for study had normal levels of IL-2-induced STAT5 phosphorylation. CONCLUSIONS: Gain-of-function mutations in STAT1 can cause an IPEX-like phenotype with normal frequency and function of Treg cells.

A novel STAT1 mutation associated with disseminated mycobacterial disease.

STAT1 is a key component of Interferon (IFN)-γ and IFN-α signaling and mediates protection against mycobacteria, fungal, viral infections, and cancer. Dominant negative inhibitory as well as gain of function heterozygous STAT1 mutations demonstrate that IFN-γ driven cellular responses need to be tightly regulated to control infections. We describe an autosomal dominant mutation in the SH2 domain of STAT1 that disrupts protein phosphorylation, c.1961T>A (M654K). The mutant allele does not permit STAT1 phosphorylation, and impairs STAT1 phosphorylation of the wild type allele. Protein dimerization is preserved but DNA binding activity, IFN-γ driven GAS-luciferase activity, and expression of IFN-γ target genes are reduced. IFN-α driven ISRE response, but not IFN-α driven GAS response, are preserved when cells are co-transfected with wild type and the mutant STAT1 constructs. M654K exerts a dominant negative effect on IFN-γ related immunity and is recessive for IFN-α induced immune function.