Genetics of Infectious and Inflammatory Diseases: Overlapping Discoveries from Association and Exome-Sequencing Studies.

Genome technologies have defined a complex genetic architecture in major infectious, inflammatory, and autoimmune disorders. High density marker arrays and Immunochips have powered genome-wide association studies (GWAS) that have mapped nearly 450 genetic risk loci in 22 major inflammatory diseases, including a core of common genes that play a central role in pathological inflammation. Whole-exome and whole-genome sequencing have identified more than 265 genes in which mutations cause primary immunodeficiencies and rare forms of severe inflammatory bowel disease. Combined analysis of inflammatory disease GWAS and primary immunodeficiencies point to shared proteins and pathways that are required for immune cell development and protection against infections and are also associated with pathological inflammation. Finally, sequencing of chromatin immunoprecipitates containing specific transcription factors, with parallel RNA sequencing, has charted epigenetic regulation of gene expression by proinflammatory transcription factors in immune cells, providing complementary information to characterize morbid genes at infectious and inflammatory disease loci.

Human T-bet Governs Innate and Innate-like Adaptive IFN-γ Immunity against Mycobacteria.

Inborn errors of human interferon gamma (IFN-γ) immunity underlie mycobacterial disease. We report a patient with mycobacterial disease due to inherited deficiency of the transcription factor T-bet. The patient has extremely low counts of circulating Mycobacterium-reactive natural killer (NK), invariant NKT (iNKT), mucosal-associated invariant T (MAIT), and Vδ2+ γδ T lymphocytes, and of Mycobacterium-non reactive classic TH1 lymphocytes, with the residual populations of these cells also producing abnormally small amounts of IFN-γ. Other lymphocyte subsets develop normally but produce low levels of IFN-γ, with the exception of CD8+ αß T and non-classic CD4+ αß TH1∗ lymphocytes, which produce IFN-γ normally in response to mycobacterial antigens. Human T-bet deficiency thus underlies mycobacterial disease by preventing the development of innate (NK) and innate-like adaptive lymphocytes (iNKT, MAIT, and Vδ2+ γδ T cells) and IFN-γ production by them, with mycobacterium-specific, IFN-γ-producing, purely adaptive CD8+ αß T, and CD4+ αß TH1∗ cells unable to compensate for this deficit.

Disruption of an antimycobacterial circuit between dendritic and helper T cells in human SPPL2a deficiency.

Human inborn errors of IFN-γ immunity underlie mycobacterial diseases. We describe patients with Mycobacterium bovis (BCG) disease who are homozygous for loss-of-function mutations of SPPL2A. This gene encodes a transmembrane protease that degrades the N-terminal fragment (NTF) of CD74 (HLA invariant chain) in antigen-presenting cells. The CD74 NTF therefore accumulates in the HLA class II+ myeloid and lymphoid cells of SPPL2a-deficient patients. This toxic fragment selectively depletes IL-12- and IL-23-producing CD1c+ conventional dendritic cells (cDC2s) and their circulating progenitors. Moreover, SPPL2a-deficient memory TH1* cells selectively fail to produce IFN-γ when stimulated with mycobacterial antigens in vitro. Finally, Sppl2a-/- mice lack cDC2s, have CD4+ T cells that produce small amounts of IFN-γ after BCG infection, and are highly susceptible to infection with BCG or Mycobacterium tuberculosis. These findings suggest that inherited SPPL2a deficiency in humans underlies mycobacterial disease by decreasing the numbers of cDC2s and impairing IFN-γ production by mycobacterium-specific memory TH1* cells.

USP15 regulates type I interferon response and is required for pathogenesis of neuroinflammation.

Genes and pathways in which inactivation dampens tissue inflammation present new opportunities for understanding the pathogenesis of common human inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis and multiple sclerosis. We identified a mutation in the gene encoding the deubiquitination enzyme USP15 (Usp15L749R) that protected mice against both experimental cerebral malaria (ECM) induced by Plasmodium berghei and experimental autoimmune encephalomyelitis (EAE). Combining immunophenotyping and RNA sequencing in brain (ECM) and spinal cord (EAE) revealed that Usp15L749R-associated resistance to neuroinflammation was linked to dampened type I interferon responses in situ. In hematopoietic cells and in resident brain cells, USP15 was coexpressed with, and functionally acted together with the E3 ubiquitin ligase TRIM25 to positively regulate type I interferon responses and to promote pathogenesis during neuroinflammation. The USP15-TRIM25 dyad might be a potential target for intervention in acute or chronic states of neuroinflammation.

Alveolar macrophage function is impaired following inhalation of berry e-cigarette vapor.

In the lower respiratory tract, the alveolar spaces are divided from the bloodstream and the external environment by only a few microns of interstitial tissue. Alveolar macrophages (AMs) defend this delicate mucosal surface from invading infections by regularly patrolling the site. AMs have three behavior modalities to achieve this goal: extending cell protrusions to probe and sample surrounding areas, squeezing the whole cell body between alveoli, and patrolling by moving the cell body around each alveolus. In this study, we found Rho GTPase, cell division control protein 42 (CDC42) expression significantly decreased after berry-flavored e-cigarette (e-cig) exposure. This shifted AM behavior from squeezing to probing. Changes in AM behavior led to a reduction in the clearance of inhaled bacteria, Pseudomonas aeruginosa. These findings shed light on pathways involved in AM migration and highlight the harmful impact of e-cig vaping on AM function.

Extracellular Vesicles: Translational Agenda Questions for Three Protozoan Parasites.

The protozoan parasites Plasmodium falciparum, Leishmania spp. and Trypanosoma cruzi continue to exert a significant toll on the disease landscape of the human population in sub-Saharan Africa and Latin America. Control measures have helped reduce the burden of their respective diseases-malaria, leishmaniasis and Chagas disease-in endemic regions. However, the need for new drugs, innovative vaccination strategies and molecular markers of disease severity and outcomes has emerged because of developing antimicrobial drug resistance, comparatively inadequate or absent vaccines, and a lack of trustworthy markers of morbid outcomes. Extracellular vesicles (EVs) have been widely reported to play a role in the biology and pathogenicity of P. falciparum, Leishmania spp. and T. cruzi ever since they were discovered. EVs are secreted by a yet to be fully understood mechanism in protozoans into the extracellular milieu and carry a cargo of diverse molecules that reflect the originator cell's metabolic state. Although our understanding of the biogenesis and function of EVs continues to deepen, the question of how EVs in P. falciparum, Leishmania spp. and T. cruzi can serve as targets for a translational agenda into clinical and public health interventions is yet to be fully explored. Here, as a consortium of protozoan researchers, we outline a plan for future researchers and pose three questions to direct an EV's translational agenda in P. falciparum, Leishmania spp. and T. cruzi. We opine that in the long term, executing this blueprint will help bridge the current unmet needs of these medically important protozoan diseases in sub-Saharan Africa and Latin America.

Chromatin-binding deubiquitinase MYSM1 acts in haematopoietic progenitors to control dendritic cell development and to program dendritic cell responses to microbial stimulation.

Dendritic cells (DCs) are the most significant antigen presenting cells of the immune system, critical for the activation of naïve T cells. The pathways controlling DC development, maturation, and effector function therefore require precise regulation to allow for an effective induction of adaptive immune response. MYSM1 is a chromatin binding deubiquitinase (DUB) and an activator of gene expression via its catalytic activity for monoubiquitinated histone H2A (H2A-K119ub), which is a highly abundant repressive epigenetic mark. MYSM1 is an important regulator of haematopoiesis in mouse and human, and a systemic constitutive loss of Mysm1 in mice results in a depletion of many haematopoietic progenitors, including DC precursors, with the downstream loss of most DC lineage cells. However, the roles of MYSM1 at the later checkpoints in DC development, maturation, activation, and effector function at present remain unknown. In the current work, using a range of novel mouse models (Mysm1flCreERT2, Mysm1flCD11c-cre, Mysm1DN), we further the understanding of MYSM1 functions in the DC lineage: assessing the requirement for MYSM1 in DC development independently of other complex developmental phenotypes, exploring its role at the later checkpoints in DC maintenance and activation in response to microbial stimulation, and testing the requirement for the DUB catalytic activity of MYSM1 in these processes. Surprisingly, we demonstrate that MYSM1 expression and catalytic activity in DCs are dispensable for the maintenance of DC numbers in vivo or for DC activation in response to microbial stimulation. In contrast, MYSM1 acts via its DUB catalytic activity specifically in haematopoietic progenitors to allow normal DC lineage development, and its loss results not only in a severe DC depletion but also in the production of functionally altered DCs, with a dysregulation of many housekeeping transcriptional programs and significantly altered responses to microbial stimulation.

Loss of MYSM1 inhibits the oncogenic activity of cMYC in B cell lymphoma.

MYSM1 is a chromatin-binding protein, widely investigated for its functions in haematopoiesis in human and mouse; however, its role in haematologic malignancies remains unexplored. Here, we investigate the cross-talk between MYSM1 and oncogenic cMYC in the transcriptional regulation of genes encoding ribosomal proteins, and the implications of these mechanisms for cMYC-driven carcinogenesis. We demonstrate that in cMYC-driven B cell lymphoma in mouse models, MYSM1-loss represses ribosomal protein gene expression and protein synthesis. Importantly, the loss of MYSM1 also strongly inhibits cMYC oncogenic activity and protects against B cell lymphoma onset and progression in the mouse models. This advances the understanding of the molecular and transcriptional mechanisms of lymphomagenesis, and suggests MYSM1 as a possible drug target for cMYC-driven malignancies.

Rocaglates as dual-targeting agents for experimental cerebral malaria.

Cerebral malaria (CM) is a severe and rapidly progressing complication of infection by Plasmodium parasites that is associated with high rates of mortality and morbidity. Treatment options are currently few, and intervention with artemisinin (Art) has limited efficacy, a problem that is compounded by the emergence of resistance to Art in Plasmodium parasites. Rocaglates are a class of natural products derived from plants of the Aglaia genus that have been shown to interfere with eukaryotic initiation factor 4A (eIF4A), ultimately blocking initiation of protein synthesis. Here, we show that the rocaglate CR-1-31B perturbs association of Plasmodium falciparum eIF4A (PfeIF4A) with RNA. CR-1-31B shows potent prophylactic and therapeutic antiplasmodial activity in vivo in mouse models of infection with Plasmodium berghei (CM) and Plasmodium chabaudi (blood-stage malaria), and can also block replication of different clinical isolates of P. falciparum in human erythrocytes infected ex vivo, including drug-resistant P. falciparum isolates. In vivo, a single dosing of CR-1-31B in P. berghei-infected animals is sufficient to provide protection against lethality. CR-1-31B is shown to dampen expression of the early proinflammatory response in myeloid cells in vitro and dampens the inflammatory response in vivo in P. berghei-infected mice. The dual activity of CR-1-31B as an antiplasmodial and as an inhibitor of the inflammatory response in myeloid cells should prove extremely valuable for therapeutic intervention in human cases of CM.

ZBTB7B (ThPOK) Is Required for Pathogenesis of Cerebral Malaria and Protection against Pulmonary Tuberculosis.

We used a genome-wide screen in N-ethyl-N-nitrosourea (ENU)-mutagenized mice to identify genes in which recessive loss-of-function mutations protect against pathological neuroinflammation. We identified an R367Q mutation in the ZBTB7B (ThPOK) protein in which homozygosity causes protection against experimental cerebral malaria (ECM) caused by infection with Plasmodium berghei ANKA. Zbtb7bR367Q homozygous mice show a defect in the lymphoid compartment expressed as severe reduction in the number of single-positive CD4 T cells in the thymus and in the periphery, reduced brain infiltration of proinflammatory leukocytes in P. berghei ANKA-infected mice, and reduced production of proinflammatory cytokines by primary T cells ex vivo and in vivo Dampening of proinflammatory immune responses in Zbtb7bR367Q mice is concomitant to increased susceptibility to infection with avirulent (Mycobacterium bovis BCG) and virulent (Mycobacterium tuberculosis H37Rv) mycobacteria. The R367Q mutation maps to the first DNA-binding zinc finger domain of ThPOK and causes loss of base contact by R367 in the major groove of the DNA, which is predicted to impair DNA binding. Global immunoprecipitation of ThPOK-containing chromatin complexes coupled to DNA sequencing (ChIP-seq) identified transcriptional networks and candidate genes likely to play key roles in CD4+ CD8+ T cell development and in the expression of lineage-specific functions of these cells. This study highlights ThPOK as a global regulator of immune function in which alterations may affect normal responses to infectious and inflammatory stimuli.

The Stat3/GR interaction code: predictive value of direct/indirect DNA recruitment for transcription outcome.

Transcription factor recruitment to genomic sites of action is primarily due to direct protein:DNA interactions. The subsequent recruitment of coregulatory complexes leads to either transcriptional activation or repression. In contrast to this canonical scheme, some transcription factors, such as the glucocorticoid receptor (GR), behave as transcriptional repressors when recruited to target genes through protein tethering. We have investigated the genome-wide prevalence of tethering between GR and Stat3 and found nonreciprocal interactions, namely that GR tethering to DNA-bound Stat3 results in transcriptional repression, whereas Stat3 tethering to GR results in synergism. Further, other schemes of GR and Stat3 corecruitment to regulatory modules result in transcriptional synergism, including neighboring and composite binding sites. The results indicate extensive transcriptional interactions between Stat3 and GR; further, they provide a genome-wide assessment of transcriptional regulation by tethering and a molecular basis for integration of signals mediated by GR and Stats in health and disease.

Deubiquitinase MYSM1 in the Hematopoietic System and beyond: A Current Review.

MYSM1 has emerged as an important regulator of hematopoietic stem cell function, blood cell production, immune response, and other aspects of mammalian physiology. It is a metalloprotease family protein with deubiquitinase catalytic activity, as well as SANT and SWIRM domains. MYSM1 normally localizes to the nucleus, where it can interact with chromatin and regulate gene expression, through deubiquitination of histone H2A and non-catalytic contacts with other transcriptional regulators. A cytosolic form of MYSM1 protein was also recently described and demonstrated to regulate signal transduction pathways of innate immunity, by promoting the deubiquitination of TRAF3, TRAF6, and RIP2. In this work we review the current knowledge on the molecular mechanisms of action of MYSM1 protein in transcriptional regulation, signal transduction, and potentially other cellular processes. The functions of MYSM1 in different cell types and aspects of mammalian physiology are also reviewed, highlighting the key checkpoints in hematopoiesis, immunity, and beyond regulated by MYSM1. Importantly, mutations in MYSM1 in human were recently linked to a rare hereditary disorder characterized by leukopenia, anemia, and other hematopoietic and developmental abnormalities. Our growing knowledge of MYSM1 functions and mechanisms of actions sheds important insights into its role in mammalian physiology and the etiology of the MYSM1-deficiency disorder in human.

Primary Immunodeficiencies and Inflammatory Disease: A Growing Genetic Intersection.

Recent advances in genome analysis have provided important insights into the genetic architecture of infectious and inflammatory diseases. The combined analysis of loci detected by genome-wide association studies (GWAS) in 22 inflammatory diseases has revealed a shared genetic core and associated biochemical pathways that play a central role in pathological inflammation. Parallel whole-exome sequencing studies have identified 265 genes mutated in primary immunodeficiencies (PID). Here, we examine the overlap between these two data sets, and find that it consists of genes essential for protection against infections and in which persistent activation causes pathological inflammation. Based on this intersection, we propose that, although strong or inactivating mutations (rare variants) in these genes may cause severe disease (PIDs), their more subtle modulation potentially by common regulatory/coding variants may contribute to chronic inflammation.

Genetic analysis of cerebral malaria in the mouse model infected with Plasmodium berghei.

Malaria is a common and sometimes fatal disease caused by infection with Plasmodium parasites. Cerebral malaria (CM) is a most severe complication of infection with Plasmodium falciparum parasites which features a complex immunopathology that includes a prominent neuroinflammation. The experimental mouse model of cerebral malaria (ECM) induced by infection with Plasmodium berghei ANKA has been used abundantly to study the role of single genes, proteins and pathways in the pathogenesis of CM, including a possible contribution to neuroinflammation. In this review, we discuss the Plasmodium berghei ANKA infection model to study human CM, and we provide a summary of all host genetic effects (mapped loci, single genes) whose role in CM pathogenesis has been assessed in this model. Taken together, the reviewed studies document the many aspects of the immune system that are required for pathological inflammation in ECM, but also identify novel avenues for potential therapeutic intervention in CM and in diseases which feature neuroinflammation.

p53 mediates loss of hematopoietic stem cell function and lymphopenia in Mysm1 deficiency.

MYSM1 is a chromatin-binding transcriptional cofactor that deubiquitinates histone H2A. Studies of Mysm1-deficient mice have shown that it is essential for hematopoietic stem cell (HSC) function and lymphopoiesis. Human carriers of a rare MYSM1-inactivating mutation display similar lymphopoietic deficiencies. However, the mechanism by which MYSM1 regulates hematopoietic homeostasis remains unclear. Here, we show that Mysm1-deficiency results in p53 protein elevation in many hematopoietic cell types. p53 is a central regulator of cellular stress responses and HSC homeostasis. We thus generated double-knockout mice to assess a potential genetic interaction between Mysm1 and p53 in hematopoiesis. Mysm1(-/-)p53(-/-) mouse characterization showed a full rescue of Mysm1(-/-) developmental and hematopoietic defects. This included restoration of lymphopoiesis, and HSC numbers and functions. These results establish p53 activation as the driving mechanism for hematopoietic abnormalities in Mysm1 deficiency. Our findings may advance the understanding of p53 regulation in hematopoiesis and implicate MYSM1 as a potential p53 cofactor.

Transcriptional mechanisms that control expression of the macrophage colony-stimulating factor receptor locus.

The proliferation, differentiation, and survival of cells of the macrophage lineage depends upon signals from the macrophage colony-stimulating factor (CSF) receptor (CSF1R). CSF1R is expressed by embryonic macrophages and induced early in adult hematopoiesis, upon commitment of multipotent progenitors to the myeloid lineage. Transcriptional activation of CSF1R requires interaction between members of the E26 transformation-specific family of transcription factors (Ets) (notably PU.1), C/EBP, RUNX, AP-1/ATF, interferon regulatory factor (IRF), STAT, KLF, REL, FUS/TLS (fused in sarcoma/ranslocated in liposarcoma) families, and conserved regulatory elements within the mouse and human CSF1R locus. One element, the Fms-intronic regulatory element (FIRE), within intron 2, is conserved functionally across all the amniotes. Lineage commitment in multipotent progenitors also requires down-regulation of specific transcription factors such as MYB, FLI1, basic leucine zipper transcriptional factor ATF-like (BATF3), GATA-1, and PAX5 that contribute to differentiation of alternative lineages and repress CSF1R transcription. Many of these transcription factors regulate each other, interact at the protein level, and are themselves downstream targets of CSF1R signaling. Control of CSF1R transcription involves feed-forward and feedback signaling in which CSF1R is both a target and a participant; and dysregulation of CSF1R expression and/or function is associated with numerous pathological conditions. In this review, we describe the regulatory network behind CSF1R expression during differentiation and development of cells of the mononuclear phagocyte system.

Mapping hyper-susceptibility to colitis-associated colorectal cancer in FVB/NJ mice.

Inbred strains of mice differ in susceptibility to colitis-associated colorectal cancer (CA-CRC). We tested 10 inbred strains of mice for their response to azoxymethane/dextran sulfate sodium-induced CA-CRC and identified a bimodal inter-strain distribution pattern when tumor multiplicity was used as a phenotypic marker of susceptibility. The FVB/NJ strain was particularly susceptible showing a higher tumor burden than any other susceptible strains (12.5-week post-treatment initiation). FVB/NJ hyper-susceptibility was detected as early as 8-week post-treatment initiation with FVB/NJ mice developing 5.5-fold more tumors than susceptible A/J or resistant B6 control mice. Linkage analysis by whole genome scan in informative (FVB/NJ×C3H/HeJ)F2 mice identified a novel susceptibility locus designated as C olon c ancer s usceptibility 6 (Ccs6) on proximal mouse chromosome 6. When gender was used as a covariate, a LOD score of 5.4 was computed with the peak marker being positioned at rs13478727, 43.8 Mbp. Mice homozygous for FVB/NJ alleles at this locus had increased tumor multiplicity compared to homozygous C3H/HeJ mice. Positional candidates in this region of chromosome 6 were analyzed with respect to a possible role in carcinogenesis and a role in inflammatory response using a new epigenetic gene scoring tool (Myeloid Inflammation Score).

Specific dysregulation of IFNγ production by natural killer cells confers susceptibility to viral infection.

Natural Killer (NK) cells contribute to the control of viral infection by directly killing target cells and mediating cytokine release. In C57BL/6 mice, the Ly49H activating NK cell receptor plays a key role in early resistance to mouse cytomegalovirus (MCMV) infection through specific recognition of the MCMV-encoded MHC class I-like molecule m157 expressed on infected cells. Here we show that transgenic expression of Ly49H failed to provide protection against MCMV infection in the naturally susceptible A/J mouse strain. Characterization of Ly49H(+) NK cells from Ly49h-A transgenic animals showed that they were able to mount a robust cytotoxic response and proliferate to high numbers during the course of infection. However, compared to NK cells from C57BL/6 mice, we observed an intrinsic defect in their ability to produce IFNγ when challenged by either m157-expressing target cells, exogenous cytokines or chemical stimulants. This effect was limited to NK cells as T cells from C57BL/6 and Ly49h-A mice produced comparable cytokine levels. Using a panel of recombinant congenic strains derived from A/J and C57BL/6 progenitors, we mapped the genetic basis of defective IFNγ production to a single 6.6 Mb genetic interval overlapping the Ifng gene on chromosome 10. Inspection of the genetic interval failed to reveal molecular differences between A/J and several mouse strains showing normal IFNγ production. The chromosome 10 locus is independent of MAPK signalling or decreased mRNA stability and linked to MCMV susceptibility. This study highlights the existence of a previously uncovered NK cell-specific cis-regulatory mechanism of Ifnγ transcript expression potentially relevant to NK cell function in health and disease.

Functional characterization of the human dendritic cell immunodeficiency associated with the IRF8(K108E) mutation.

We have previously reported on a unique patient in whom homozygosity for a mutation at IRF8 (IRF8(K108E)) causes a severe immunodeficiency. Laboratory evaluation revealed a highly unusual myeloid compartment, remarkable for the complete absence of CD141 and CD161 monocytes, absence of CD11c1 conventional dendritic cells (DCs) and CD11c1/CD1231 plasmacytoid DCs, and striking granulocytic hyperplasia. The patient initially presented with severe disseminated mycobacterial and mucocutaneous fungal infections and was ultimately cured by cord blood transplant. Sequencing RNA from the IRF8(K108E) patient's primary blood cells prior to transplant shows not only depletion of IRF8-bound and IRF8-regulated transcriptional targets, in keeping with the distorted composition of the myeloid compartment, but also a paucity of transcripts associated with activated CD41 and CD81 T lymphocytes. This suggests that T cells reared in the absence of a functional antigen-presenting compartment in IRF8(K108E) are anergic. Biochemical characterization of the IRF8(K108E) mutant in vitro shows that loss of the positively charged side chain at K108 causes loss of nuclear localization and loss of transcriptional activity, which is concomitant with decreased protein stability, increased ubiquitination, increased small ubiquitin-like modification, and enhanced proteasomal degradation. These findings provide functional insight into the molecular basis of immunodeficiency associated with loss of IRF8.