Anna Rydström

Early cellular responses to Salmonella infection: dendritic cells, monocytes, and more

Summary: Dendritic cells (DCs), monocytes, macrophages, and neutrophils are myeloid‐derived phagocytes critical to controlling bacterial infections, and these cells have complementary functions to ensure host survival. Recent data have shed light on the dynamics and function of myeloid cells at the early stage of infection. In particular, murine infection models with Salmonella enterica serovar Typhimurium have been useful for understanding the host response required to develop immunity to systemic salmonellosis. This review summarizes the early cellular responses in the intestinal lymphoid tissues to Salmonella and discusses Peyers patch‐dependent and ‐independent penetration of bacteria through the intestinal epithelium. Once Salmonella accesses host tissue, phagocytes respond by recruitment, redistribution, and activation in intestinal tissues. Recruited monocytes are specialized in controlling bacterial replication by producing anti‐microbial molecules but are poor antigen‐presenting cells. In contrast, DCs undergo maturation by direct (bacteria‐mediated) and indirect (cytokine‐mediated) pathways in vivo to optimize their antigen presentation capacity, and directly matured DCs have unique mechanisms to ensure T‐cell stimulation. Toll‐like receptor signaling is critical to DC maturation and myeloid cell recruitment during Salmonella infection, and the role of myeloid differentiation factor 88 (MyD88)‐dependent and MyD88‐independent pathways as well as proinflammatory cytokines and type 1 interferons in these processes are discussed.

Monocyte Recruitment, Activation, and Function in the Gut-Associated Lymphoid Tissue during Oral Salmonella Infection

Neutrophils, monocytes, and dendritic cells (DC) are phenotypically and functionally related phagocytes whose presence in infected tissues is critical to host survival. Their overlapping expression pattern of surface molecules, the differentiation capacity of monocytes, and the presence of monocyte subsets underscores the complexity of understanding the role of these cells during infection. In this study we use five- to seven-color flow cytometry to assess the phenotype and function of monocytes recruited to Peyer’s patches (PP) and mesenteric lymph nodes (MLN) after oral Salmonella infection of mice. The data show that CD68highGr-1int (intermediate) monocytes, along with CD68intGr-1high neutrophils, rapidly accumulate in PP and MLN. The monocytes have increased MHC-II and costimulatory molecule expression and, in contrast to neutrophils and DC, produce inducible NO synthase. Although neutrophils and monocytes from infected mice produce TNF-α and IL-1β upon ex vivo culture, DC do not. In addition, although recruited monocytes internalize Salmonella in vitro and in vivo they did not induce the proliferation of OT-II CD4+ T cells after coincubation with Salmonella expressing OVA despite their ability to activate OT-II cells when pulsed with the OVA323–339 peptide. We also show that recruited monocytes enter the PP of infected mice independently of the mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Finally, recruited but not resident monocytes increase in the blood of orally infected mice, and MHC-II up-regulation, but not TNF-α or iNOS production, occur already in the blood. These studies are the first to describe the accumulation and function of monocyte subsets in the blood and GALT during oral Salmonella infection.

Immunity to Salmonella from a dendritic point of view

Dendritic cells (DC) are the key link between innate and adaptive immunity. Features of DC, including their presence at sites of antigen entry, their ability to migrate from peripheral sites to secondary lymphoid organs, and their superior capacity to stimulate naïve T cells places them in this pivotal role in the immune system. DC also produce cytokines, particularly IL‐12, upon antigen encounter and can thus influence the ensuing adaptive immune response. As DC are phagocytic antigen‐presenting cells located at sites exposed to bacterial invaders, studies have been performed to gain insight into the role of DC in combating bacterial infections. Indeed, studies with Salmonella have shown that DC can internalize and process this bacterium for peptide presentation on MHC‐II as well as MHC‐I. DC can also act as bystander antigen‐presenting cells by presenting Salmonella antigens after internalizing neighbouring cells that have undergone Salmonella‐induced apoptotic death. DC also produce IL‐12 and TNF‐α upon Salmonella encounter. Moreover, studies in a murine infection model have shown that splenic DC increase surface expression of co‐stimulatory molecules during infection, and DC contain intracellular bacteria. In addition, quantitative changes occur in splenic DC numbers in the early stages of oral Salmonella infection, and this is accompanied by redistribution of the defined DC subsets in the spleen of infected mice. DC from Salmonella‐infected mice also produce cytokines and can stimulate bacteria‐specific T cells upon ex vivo co‐culture. In addition, DC may play a role in the traversal of bacteria from the intestinal lumen. Studying the function of DC during Salmonella infection provides insight into the capacity of this sophisticated antigen‐presenting cell to initiate and modulate the immune response to bacteria.

Spontaneous colitis in Muc2-deficient mice reflects clinical and cellular features of active ulcerative colitis.

Background The colonic mucus layer plays a critical role in intestinal homeostasis by limiting contact between luminal bacteria and the mucosal immune system. A defective mucus barrier in animal models allows bacterial contact with the intestinal epithelium and results in spontaneous colitis. A defective mucus barrier is also a key feature of active ulcerative colitis (UC). Alterations in the immune compartment due to intestinal bacterial breach in mice lacking the colon mucus barrier have not been characterized and correlated to active UC. Aims To characterize alterations in the immune compartment due to intestinal bacterial breach in Muc2−/− mice, which lack the colon mucus barrier, and correlate the findings to active UC. Methods Bacterial contact with colon epithelium and penetration into colon tissue was examined in Muc2−/− mice and colon biopsies from patients with active UC using fluorescence microscopy and qPCR. Neutrophils, lymphocytes, CD103+ dendritic cell subsets and macrophages in colon from Muc2−/− mice and biopsies from UC patients were quantitated by flow cytometry. Results Inflamed UC patients and Muc2−/− mice had bacteria in contact with the colon epithelium. Bacterial rRNA was present in colonic mucosa in humans and Muc2−/− mice and in the draining lymph nodes of mice. Inflamed Muc2−/− mice and UC patients had elevated colon neutrophils, T cells and macrophages while a reduced frequency of CD103+ DCs was present in the inflamed colon of both mice and humans. Conclusions The parallel features of the colon immune cell compartment in Muc2−/− mice and UC patients supports the usefulness of this model to understand the early phase of spontaneous colitis and will provide insight into novel strategies to treat UC.

Monocyte and neutrophil recruitment during oral Salmonella infection is driven by MyD88‐derived chemokines

Oral Salmonella infection recruits phagocytes to Peyers patches (PP) and MLN. The chemokines induced in infected PP and MLN, the cellular sources during infection and the TLR signaling pathways involved in vivo are not known. Here, we show that CCL2, CXCL9 and CXCL2 mRNA are up‐regulated in PP and MLN coincident with the first arrival of monocytes and neutrophils. Laser capture microdissection microscopy revealed that chemokine mRNA up‐regulation was differently distributed in PP. Despite this, recruited monocytes and neutrophils formed inflammatory cell clusters throughout PP. Monocytes and neutrophils purified from infected mice preferentially produced CXCL2 and small amounts of CCL2, and neutrophils from infected mice migrated towards CXCL2 and CCL3. Furthermore, phagocyte recruitment to PP and MLN was intact in mice lacking TLR4 alone and when signaling through TLR4 and TLR5 was simultaneously absent; however, recruitment was compromised in MyD88−/− and more so in MyD88−/−TLR4−/− double knockout mice. Phagocyte release into the blood, however, was only marginally reduced in MyD88−/−TLR4−/− mice. Defective phagocyte recruitment to PP and MLN of MyD88−/−TLR4−/− mice was paralleled by low chemokine induction. These data provide insight into the chemokines and TLR signaling pathways that orchestrate the early phagocyte response to oral Salmonella infection.

Role of natural killer cell subsets and natural cytotoxicity receptors for the outcome of immunotherapy in acute myeloid leukemia.

In a phase IV trial, 84 patients (age 18–79) with acute myeloid leukemia (AML) in first complete remission (CR) received cycles of immunotherapy with histamine dihydrochloride (HDC) and low-dose human recombinant interleukin 2 (IL-2) for 18 months to prevent leukemic relapse. During cycles, the treatment resulted in expansion of CD56bright (CD3−/16−/56bright) and CD16+ (CD3−/16+/56+) natural killer (NK) cells in the blood along with increased NK cell expression of the natural cytotoxicity receptors (NCRs) NKp30 and NKp46. Multivariate analyses correcting for age and risk group demonstrated that high CD56bright NK cell counts and high expression of NKp30 or NKp46 on CD16+ NK cells independently predicted leukemia-free survival (LFS) and overall survival (OS). Our results suggest that the dynamics of NK cell subsets and their NCR expression may determine the efficiency of relapse-preventive immunotherapy in AML.

Redox Remodeling by Dendritic Cells Protects Antigen-Specific T Cells against Oxidative Stress

Microorganisms and microbial products induce the release of reactive oxygen species (ROS) from monocytes and other myeloid cells, which may trigger dysfunction and apoptosis of adjacent lymphocytes. Therefore, T cell-mediated immunity is likely to comprise mechanisms of T cell protection against ROS-inflicted toxicity. The present study aimed to clarify the dynamics of reduced sulfhydryl groups (thiols) in human T cells after presentation of viral and bacterial Ags by dendritic cells (DCs) or B cells. DCs, but not B cells, efficiently triggered intra- and extracellular thiol expression in T cells with corresponding Ag specificity. After interaction with DCs, the Ag-specific T cells acquired the capacity to neutralize exogenous oxygen radicals and resisted ROS-induced apoptosis. Our results imply that DCs provide Ag-specific T cells with antioxidative thiols during Ag presentation, which suggests a novel aspect of DC/T cell cross-talk of relevance to the maintenance of specific immunity in inflamed or infected tissue.

NK cell expression of natural cytotoxicity receptors may determine relapse risk in older AML patients undergoing immunotherapy for remission maintenance

In a phase IV trial, eighty-four patients (age 18–79) with acute myeloid leukemia (AML) in first complete remission (CR) received cycles of immunotherapy with histamine dihydrochloride (HDC) and low-dose human recombinant interleukin-2 (IL-2) to prevent relapse in the post-consolidation phase. Aspects of natural killer (NK) cell biology were analyzed before and during immunotherapy with focus on outcome in older patients. In younger (<60 years old, n = 37) and older patients (>60 years old, n = 47), treatment with HDC/IL-2 resulted in an expansion of CD56bright and CD16+ NK cells in blood along with an increased NK cell expression of the natural cytotoxicity receptors (NCR) NKp30 and NKp46. In older patients, a high expression of NKp30 or NKp46 on CD16+ NK cells before and during therapy predicted leukemia-free and overall survival. These results suggest that NK cell functions determine relapse risk and survival in older AML patients and point to biomarkers of efficacy in protocols for remission maintenance.

Salmonella inhibits monocyte differentiation into CD11chiMHC-IIhi cells in a MyD88-dependent fashion

Monocytes and DCs originate from a shared precursor in the bone marrow, and steady‐state DCs in lymphoid organs develop directly from the precursor rather than via a monocyte intermediate. However, monocytes can differentiate into DCs in tissues such as the lung and gut mucosa and into macrophages in most tissues. As Ly6Chi monocytes accumulate in lymphoid organs during oral Salmonella infection, we investigated their ability to develop into potential DCs, identified as CD11chiMHC‐IIhi cells, in infected hosts. Ly6Chi monocytes, isolated from the blood of Salmonella‐infected mice, developed into CD11chiMHC‐IIhi cells after culture with GM‐CSF or Flt3L. In contrast, the same monocytes cultured in the presence of GM‐CSF and heat‐killed Salmonella did not differentiate into CD11chiMHC‐IIhi cells. The bacteria‐induced differentiation block was dependent on TLRs, as monocytes from MyD88−/− mice converted into CD11chiMHC‐IIhi cells even in the presence of bacteria. We hypothesized that Salmonella‐activated wild‐type monocytes secreted mediators that inhibited differentiation of MyD88−/−‐derived monocytes. However, IL‐6, IL‐10, TNF‐α, or IL‐12p70 did not account for the inhibition. Finally, monocyte‐derived CD11chiMHC‐IIhi cells pulsed with OVA peptide or protein did not induce proliferation of antigen‐specific CD4+ T cells but rather, suppressed the ability of DCs to activate CD4+ T cells. Overall, the data show that Ly6Chi monocytes from Salmonella‐infected mice develop into CD11chiMHC‐IIhi cells with poor antigen‐presentation capacity when cultured ex vivo, and that monocyte exposure to Salmonella inhibits their differentiation into CD11chiMHC‐IIhi cells in a MyD88‐dependent fashion.

Role of regulatory T cells in acute myeloid leukemia patients undergoing relapse-preventive immunotherapy

Regulatory T cells (Tregs) have been proposed to dampen functions of anti-neoplastic immune cells and thus promote cancer progression. In a phase IV trial (Re:Mission Trial, NCT01347996, http://www.clinicaltrials.gov) 84 patients (age 18–79) with acute myeloid leukemia (AML) in first complete remission (CR) received ten consecutive 3-week cycles of immunotherapy with histamine dihydrochloride (HDC) and low-dose interleukin-2 (IL-2) to prevent relapse of leukemia in the post-consolidation phase. This study aimed at defining the features, function and dynamics of Foxp3+CD25highCD4+ Tregs during immunotherapy and to determine the potential impact of Tregs on relapse risk and survival. We observed a pronounced increase in Treg counts in peripheral blood during initial cycles of HDC/IL-2. The accumulating Tregs resembled thymic-derived natural Tregs (nTregs), showed augmented expression of CTLA-4 and suppressed the cell cycle proliferation of conventional T cells ex vivo. Relapse of AML was not prognosticated by Treg counts at onset of treatment or after the first cycle of immunotherapy. However, the magnitude of Treg induction was diminished in subsequent treatment cycles. Exploratory analyses implied that a reduced expansion of Tregs in later treatment cycles and a short Treg telomere length were significantly associated with a favorable clinical outcome. Our results suggest that immunotherapy with HDC/IL-2 in AML entails induction of immunosuppressive Tregs that may be targeted for improved anti-leukemic efficiency.