Macrophages in the synovial lining niche initiate neutrophil recruitment and articular inflammation.

The first immune-activating changes within joint resident cells that lead to pathogenic leukocyte recruitment during articular inflammation remain largely unknown. In this study, we employ state-of-the-art confocal microscopy and image analysis in a systemic, whole-organ, and quantitative way to present evidence that synovial inflammation begins with the activation of lining macrophages. We show that lining, but not sublining macrophages phagocytose immune complexes containing the model antigen. Using the antigen-induced arthritis (AIA) model, we demonstrate that on recognition of antigen-antibody complexes, lining macrophages undergo significant activation, which is dependent on interferon regulatory factor 5 (IRF5), and produce chemokines, most notably CXCL1. Consequently, at the onset of inflammation, neutrophils are preferentially recruited in the vicinity of antigen-laden macrophages in the synovial lining niche. As inflammation progresses, neutrophils disperse across the whole synovium and form swarms in synovial sublining during resolution. Our study alters the paradigm of lining macrophages as immunosuppressive cells to important instigators of synovial inflammation.

Defactinib inhibits PYK2 phosphorylation of IRF5 and reduces intestinal inflammation.

Interferon regulating factor 5 (IRF5) is a multifunctional regulator of immune responses, and has a key pathogenic function in gut inflammation, but how IRF5 is modulated is still unclear. Having performed a kinase inhibitor library screening in macrophages, here we identify protein-tyrosine kinase 2-beta (PTK2B/PYK2) as a putative IRF5 kinase. PYK2-deficient macrophages display impaired endogenous IRF5 activation, leading to reduction of inflammatory gene expression. Meanwhile, a PYK2 inhibitor, defactinib, has a similar effect on IRF5 activation in vitro, and induces a transcriptomic signature in macrophages similar to that caused by IRF5 deficiency. Finally, defactinib reduces pro-inflammatory cytokines in human colon biopsies from patients with ulcerative colitis, as well as in a mouse colitis model. Our results thus implicate a function of PYK2 in regulating the inflammatory response in the gut via the IRF5 innate sensing pathway, thereby opening opportunities for related therapeutic interventions for inflammatory bowel diseases and other inflammatory conditions.

Intercrypt sentinel macrophages tune antibacterial NF-κB responses in gut epithelial cells via TNF.

Intestinal epithelial cell (IEC) NF-κB signaling regulates the balance between mucosal homeostasis and inflammation. It is not fully understood which signals tune this balance and how bacterial exposure elicits the process. Pure LPS induces epithelial NF-κB activation in vivo. However, we found that in mice, IECs do not respond directly to LPS. Instead, tissue-resident lamina propria intercrypt macrophages sense LPS via TLR4 and rapidly secrete TNF to elicit epithelial NF-κB signaling in their immediate neighborhood. This response pattern is relevant also during oral enteropathogen infection. The macrophage-TNF-IEC axis avoids responses to luminal microbiota LPS but enables crypt- or tissue-scale epithelial NF-κB responses in proportion to the microbial threat. Thereby, intercrypt macrophages fulfill important sentinel functions as first responders to Gram-negative microbes breaching the epithelial barrier. The tunability of this crypt response allows the induction of defense mechanisms at an appropriate scale according to the localization and intensity of microbial triggers.

Regional specialization of macrophages along the gastrointestinal tract.

The tissue microenvironment is a major driver in imprinting tissue-specific macrophage functions in various mammalian tissues. As monocytes are recruited into the gastrointestinal (GI) tract at steady state and inflammation, they rapidly adopt a tissue-specific and distinct transcriptome. However, the GI tract varies significantly along its length, yet most studies of intestinal macrophages do not directly compare the phenotype and function of these macrophages in the small and large intestine, thus leading to disparities in data interpretations. This review highlights differences along the GI tract that are likely to influence macrophage function, with a specific focus on diet and microbiota. This analysis may fuel further investigation regarding the interplay between the intestinal immune system and GI tissue microenvironments, ideally providing unique therapeutic targets to modulate specific intestinal macrophage populations and/or functions.

IRF5 guides monocytes toward an inflammatory CD11c+ macrophage phenotype and promotes intestinal inflammation.

Mononuclear phagocytes (MNPs) are vital for maintaining intestinal homeostasis but, in response to acute microbial stimulation, can also trigger immunopathology, accelerating recruitment of Ly6Chi monocytes to the gut. The regulators that control monocyte tissue adaptation in the gut remain poorly understood. Interferon regulatory factor 5 (IRF5) is a transcription factor previously shown to play a key role in maintaining the inflammatory phenotype of macrophages. Here, we investigate the impact of IRF5 on the MNP system and physiology of the gut at homeostasis and during inflammation. We demonstrate that IRF5 deficiency has a limited impact on colon physiology at steady state but ameliorates immunopathology during Helicobacter hepaticus-induced colitis. Inhibition of IRF5 activity in MNPs phenocopies global IRF5 deficiency. Using a combination of bone marrow chimera and single-cell RNA-sequencing approaches, we examined the intrinsic role of IRF5 in controlling colonic MNP development. We demonstrate that IRF5 promotes differentiation of Ly6Chi monocytes into CD11c+ macrophages and controls the production of antimicrobial and inflammatory mediators by these cells. Thus, we identify IRF5 as a key transcriptional regulator of the colonic MNP system during intestinal inflammation.

Intestinal epithelial NAIP/NLRC4 restricts systemic dissemination of the adapted pathogen Salmonella Typhimurium due to site-specific bacterial PAMP expression.

Inflammasomes can prevent systemic dissemination of enteropathogenic bacteria. As adapted pathogens including Salmonella Typhimurium (S. Tm) have evolved evasion strategies, it has remained unclear when and where inflammasomes restrict their dissemination. Bacterial population dynamics establish that the NAIP/NLRC4 inflammasome specifically restricts S. Tm migration from the gut to draining lymph nodes. This is solely attributable to NAIP/NLRC4 within intestinal epithelial cells (IECs), while S. Tm evades restriction by phagocyte NAIP/NLRC4. NLRP3 and Caspase-11 also fail to restrict S. Tm mucosa traversal, migration to lymph nodes, and systemic pathogen growth. The ability of IECs (not phagocytes) to mount a NAIP/NLRC4 defense in vivo is explained by particularly high NAIP/NLRC4 expression in IECs and the necessity for epithelium-invading S. Tm to express the NAIP1-6 ligands-flagella and type-III-secretion-system-1. Imaging reveals both ligands to be promptly downregulated following IEC-traversal. These results highlight the importance of intestinal epithelial NAIP/NLRC4 in blocking bacterial dissemination in vivo, and explain why this constitutes a uniquely evasion-proof defense against the adapted enteropathogen S. Tm.

Escherichia coli limits Salmonella Typhimurium infections after diet shifts and fat-mediated microbiota perturbation in mice.

The microbiota confers colonization resistance, which blocks Salmonella gut colonization1. As diet affects microbiota composition, we studied whether food composition shifts enhance susceptibility to infection. Shifting mice to diets with reduced fibre or elevated fat content for 24 h boosted Salmonella Typhimurium or Escherichia coli gut colonization and plasmid transfer. Here, we studied the effect of dietary fat. Colonization resistance was restored within 48 h of return to maintenance diet. Salmonella gut colonization was also boosted by two oral doses of oleic acid or bile salts. These pathogen blooms required Salmonella's AcrAB/TolC-dependent bile resistance. Our data indicate that fat-elicited bile promoted Salmonella gut colonization. Both E. coli and Salmonella show much higher bile resistance than the microbiota. Correspondingly, competitive E. coli can be protective in the fat-challenged gut. Diet shifts and fat-elicited bile promote S. Typhimurium gut infections in mice lacking E. coli in their microbiota. This mouse model may be useful for studying pathogen-microbiota-host interactions, the protective effect of E. coli, to analyse the spread of resistance plasmids and assess the impact of food components on the infection process.

Development of a Novel CD4+ TCR Transgenic Line That Reveals a Dominant Role for CD8+ Dendritic Cells and CD40 Signaling in the Generation of Helper and CTL Responses to Blood-Stage Malaria.

We describe an MHC class II (I-Ab)-restricted TCR transgenic mouse line that produces CD4+ T cells specific for Plasmodium species. This line, termed PbT-II, was derived from a CD4+ T cell hybridoma generated to blood-stage Plasmodium berghei ANKA (PbA). PbT-II cells responded to all Plasmodium species and stages tested so far, including rodent (PbA, P. berghei NK65, Plasmodium chabaudi AS, and Plasmodium yoelii 17XNL) and human (Plasmodium falciparum) blood-stage parasites as well as irradiated PbA sporozoites. PbT-II cells can provide help for generation of Ab to P. chabaudi infection and can control this otherwise lethal infection in CD40L-deficient mice. PbT-II cells can also provide help for development of CD8+ T cell-mediated experimental cerebral malaria (ECM) during PbA infection. Using PbT-II CD4+ T cells and the previously described PbT-I CD8+ T cells, we determined the dendritic cell (DC) subsets responsible for immunity to PbA blood-stage infection. CD8+ DC (a subset of XCR1+ DC) were the major APC responsible for activation of both T cell subsets, although other DC also contributed to CD4+ T cell responses. Depletion of CD8+ DC at the beginning of infection prevented ECM development and impaired both Th1 and follicular Th cell responses; in contrast, late depletion did not affect ECM. This study describes a novel and versatile tool for examining CD4+ T cell immunity during malaria and provides evidence that CD4+ T cell help, acting via CD40L signaling, can promote immunity or pathology to blood-stage malaria largely through Ag presentation by CD8+ DC.