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IntroductionEarly sepsis is a life-threatening immune dysregulation believed to feature a “cytokine storm” due to activation of pattern recognition receptors by pathogen and danger associated molecular patterns. However, treatments with single toll-like receptor (TLR) blockers have shown no clinical benefit. We speculated that sepsis patients at the time of diagnosis are heterogeneous in relation to their cytokine production and its potential inhibition by a triple cocktail of TLR blockers. Accordingly, we analyzed inflammatory cytokine production in whole blood assays from early sepsis patients and determined the effects of triple TLR-blockade.MethodsWhole blood of 51 intensive care patients sampled within 24h of meeting Sepsis-3 criteria was incubated for 6h without or with specific TLR2, 4, and 7/8 stimuli or suspensions of heat-killed S. aureus or E. coli bacteria as pan-TLR challenges, and also with a combination of monoclonal antibodies against TLR2 and 4 and chloroquine (endosomal TLR inhibition), subsequent to dose optimization. Concentrations of tumor necrosis factor (TNF), Interleukin(IL)-6, IL-8, IL-10, IL-1α and IL-1β were measured (multiplex ELISA) before and after incubation. Samples from 11 sex and age-matched healthy volunteers served as controls and for dose-finding studies.ResultsOnly a fraction of sepsis patient samples revealed ongoing cytokine production ex vivo despite sampling within 24 h of first meeting Sepsis-3 criteria. In dose finding studies, inhibition of TLR2, 4 and endosomal TLRs reliably suppressed cytokine production to specific TLR agonists and added bacteria. However, inflammatory cytokine production ex vivo was only suppressed in the high cytokine producing samples but not in the majority. The suppressive response to TLR-blockade correlated both with intraassay inflammatory cytokine production (r=0.29–0.68; p

Clinical and experimental studies indicate that the bacterial and fungal gut microbiota modulates immune responses in distant organs including the lungs. Immune dysregulation is associated with severe SARS-CoV-2 infection, and several groups have observed gut bacterial dysbiosis in SARS-CoV-2 infected patients, while the fungal gut microbiota remains poorly defined in these patients. We analyzed the fungal gut microbiome from rectal swabs taken prior to anti-infective treatment in 30 SARS-CoV-2 positive (21 non-severe COVID-19 and 9 developing severe/critical COVID-19 patients) and 23 SARS-CoV-2 negative patients by ITS2-sequencing. Pronounced but distinct interconnected fungal communities distinguished SARS-CoV-2 positive and negative patients. Fungal gut microbiota in severe/critical COVID-19 illness was characterized by a reduced diversity, richness and evenness and by an increase of the relative abundance of the Ascomycota phylum compared with non-severe COVID-19 illness. A dominance of a single fungal species with a relative abundance of >75% was a frequent feature in severe/critical COVID-19. The dominating fungal species were highly variable between patients even within the groups. Several fungal taxa were depleted in patients with severe/critical COVID-19.The distinct compositional changes of the fungal gut microbiome in SARS-CoV-2 infection, especially in severe COVID-19 illness, illuminate the necessity of a broader approach to investigate whether the differences in the fungal gut microbiome are consequences of SARS-CoV-2 infection or a predisposing factor for critical illness.

The gut microbiota contributes to maintaining human health and regulating immune responses. Severe COVID-19 illness is associated with a dysregulated pro-inflammatory immune response. The effect of SARS-CoV-2 on altering the gut microbiome and the relevance of the gut microbiome on COVID-19 severity needs to be clarified. In this prospective study, we analyzed the gut microbiome of 212 patients of a tertiary care hospital (117 patients infected with SARS-CoV-2 and 95 SARS-CoV-2 negative patients) using 16S rRNA gene sequencing of the V3-V4 region. Inflammatory markers and immune cells were quantified from blood. The gut microbiome in SARS-CoV-2 infected patients was characterized by a lower bacterial richness and distinct differences in the gut microbiome composition, including an enrichment of the phyla Proteobacteria and Bacteroidetes and a decrease of Actinobacteria compared to SARS-CoV-2 negative patients. The relative abundance of several genera including Bifidobacterium, Streptococcus and Collinsella was lower in SARS-CoV-2 positive patients while the abundance of Bacteroides and Enterobacteriaceae was increased. Higher pro-inflammatory blood markers and a lower CD8+ T cell number characterized patients with severe COVID-19 illness. The gut microbiome of patients with severe/critical COVID-19 exhibited a lower abundance of butyrate-producing genera Faecalibacterium and Roseburia and a reduction in the connectivity of a distinct network of anti-inflammatory genera that was observed in patients with mild COVID-19 illness and in SARS-CoV-2 negative patients. Dysbiosis of the gut microbiome associated with a pro-inflammatory signature may contribute to the hyperinflammatory immune response characterizing severe COVID-19 illness.

Patients suffering from ulcerative colitis are at increased risk of developing colorectal cancer. Although the exact underlying mechanisms of inflammation-associated carcinogenesis remain unknown, the intestinal microbiota as well as pathogenic bacteria are discussed as contributors to inflammation and colitis-associated colon cancer (CAC). In the present study, we analyzed the impact of TLR4, the receptor for Gram-negative bacteria derived lipopolysaccharides, on intestinal inflammation and tumorigenesis in a murine model of CAC. During the inflammatory phases of CAC development, we observed a strong upregulation of Tlr4 expression in colonic tissues. Blocking of TLR4 signaling by a small-molecule-specific inhibitor during the inflammatory phases of CAC strongly diminished the development and progression of colonic tumors, which was accompanied by decreased numbers of infiltrating macrophages and reduced colonic pro-inflammatory cytokine levels compared to CAC control mice. Interestingly, inhibiting bacterial signaling by antibiotic treatment during the inflammatory phases of CAC also protected mice from severe intestinal inflammation and almost completely prevented tumor growth. Nevertheless, application of antibiotics involved rapid and severe body weight loss and might have unwanted side effects. Our results indicate that bacterial activation of TLR4 on innate immune cells in the colon triggers inflammation and promotes tumor growth. Thus, the inhibition of the TLR4 signaling during intestinal inflammation might be a novel approach to impede CAC development.

The composition of the oral milieu reflects oral health. Saliva provides an environment for multiple microorganisms, and contains soluble factors and immune cells. Neutrophils, which rapidly react on the changes in the microenvironment, are a major immune cell population in saliva and thus may serve as a biomarker for oral pathologies. This review focuses on salivary neutrophils in the oral cavity, their phenotype changes in physiological and pathological conditions, as well as on factors regulating oral neutrophil amount, activation and functionality, with special emphasis on oral cancer and its risk factors.

Research into the cooperative pathogenicity of microbes in cystic fibrosis (CF) lungs is crucial for an understanding of the pathophysiology of infections and the development of novel treatment strategies. This study investigated the impact of the common CF-associated bacterial pathogen Pseudomonas aeruginosa on the black yeast Exophiala dermatitidis. It evaluated the planktonic growth, biofilm formation, morphology, and virulence of the fungus in the presence or absence of P. aeruginosa. It also determined the role of P. aeruginosa quorum-sensing (QS) molecules within these interactions, e.g., by using sterile culture filtrate and QS-deficient mutants. P. aeruginosa is known to inhibit the planktonic growth of E. dermatitidis. We found that fungal biofilm formation increased in the presence of P. aeruginosa after 24 h but is decreased significantly after 48 h. This effect was reversed when, instead of QS wild-type strains, ΔlasR, and ΔrhlR mutants were added to E. dermatitidis biofilm formation. The number and length of hyphae were substantially reduced when E. dermatitidis was co-cultivated with P. aeruginosa, but not when it was co-cultivated with the mutants. Experiments testing the virulence of E. dermatitidis in the greater wax moth Galleria mellonella showed a synergetic effect on larval killing when E. dermatitidis was injected together with P. aeruginosa culture filtrate. Survival rates were decreased when biofilm culture filtrate was added but not when planktonic culture filtrate was added. In summary, P. aeruginosa affects the growth, morphology, biofilm formation, and virulence of E. dermatitidis. N-acyl-L-homoserine lactone (AHL) QS molecules regulated factors that have been shown to contribute to the inhibition of the ability of E. dermatitidis to form filaments and biofilm.

Inflammatory diseases of the gastrointestinal tract are emerging as a global problem with increased evidence and prevalence in numerous countries. A dysregulated sphingolipid metabolism occurs in patients with ulcerative colitis and is discussed to contribute to its pathogenesis. In the present study, we determined the impact of acid sphingomyelinase (Asm), which catalyzes the hydrolysis of sphingomyelin to ceramide, on the course of Citrobacter (C.) rodentium-driven colitis. C. rodentium is an enteric pathogen and induces colonic inflammation very similar to the pathology in patients with ulcerative colitis. We found that mice with Asm deficiency or Asm inhibition were strongly susceptible to C. rodentium infection. These mice showed increased levels of C. rodentium in the feces and were prone to bacterial spreading to the systemic organs. In addition, mice lacking Asm activity showed an uncontrolled inflammatory Th1 and Th17 response, which was accompanied by a stronger colonic pathology compared to infected wild type mice. These findings identified Asm as an essential regulator of mucosal immunity to the enteric pathogen C. rodentium.

The enzyme acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and is thereby involved in several cellular processes such as differentiation, proliferation, and apoptosis in different cell types. However, the function of ASM in T cells is still not well characterized. Here, we used T cell-specific ASM overexpressing mice (t-ASM/CD4cre) to clarify the impact of cell-intrinsic ASM activity on T cell function in vitro and in vivo. We showed that t-ASM/CD4cre mice exhibit decreased frequencies of Foxp3+ T regulatory cells (Tregs) within the spleen. Enforced T cell-specific ASM expression resulted in less efficient induction of Tregs and promoted differentiation of CD4+CD25− naïve T cells into IFN-γ producing Th1 cells in vitro. Further analysis revealed that ASM-overexpressing T cells from t-ASM/CD4cre mice show elevated T cell receptor (TCR) signaling activity accompanied with increased proliferation upon stimulation in vitro. Plasmodium yoelii infection of t-ASM/CD4cre mice resulted in enhanced T cell activation and was associated with reduced parasitemia in comparison to infected control mice. Hence, our results provide evidence that ASM activity modulates T cell function in vitro and in vivo.

T cell dysfunction and immunosuppression are characteristic for chronic viral infections and contribute to viral persistence. Overcoming these burdens is the goal of new therapeutic strategies to cure chronic infectious diseases. We recently described that therapeutic vaccination of chronic retrovirus infected mice with a calcium phosphate (CaP) nanoparticle (NP)-based vaccine carrier, functionalized with CpG and viral peptides is able to efficiently reactivate the CD8+ T cell response and improve the eradication of virus infected cells. However, the mechanisms underlying this effect were largely unclear. While type I interferons (IFNs I) are considered to drive T cell exhaustion by persistent immune activation during chronic viral infection, we here describe an indispensable role of IFN I induced by therapeutic vaccination to efficiently reinforce cytotoxic CD8+ T cells (CTL) and improve control of chronic retroviral infection. The induction of IFN I is CpG dependent and leads to significant IFN signaling indicated by upregulation of IFN stimulated genes. By vaccinating chronically retrovirus-infected mice lacking the IFN I receptor (IFNAR−/−) or by blocking IFN I signaling in vivo during therapeutic vaccination, we demonstrate that IFN I signaling is necessary to drive full reactivation of CTLs. Surprisingly, we also identified an impaired suppressive capability of regulatory T cells in the presence of IFNα, which implicates an important role for vaccine-induced IFNα in the regulation of the T cell response during chronic retroviral infection. Our data suggest that inducing IFN I signaling in conjunction with the presentation of viral antigens can reactivate immune functions and reduce viral loads in chronic infections. Therefore, we propose CaP NPs as potential therapeutic tool to treat chronic infections.

Dendritic cells (DCs) are highly specialized antigen-presenting cells that orchestrate adaptive immune responses to pathogens. During malaria infection pro- and anti-inflammatory T cell responses have to be tightly balanced to ensure parasite clearance without induction of severe immune pathologies. However, the precise role of CD11chigh DCs in this process is still discussed controversially. Here, we demonstrate that long-term depletion of conventional CD11chigh DCs in Plasmodium yoelii (P. yoelii)-infected diphtheria toxin (DT)-treated RosaiDTR/CD11c-cre mice interferes with the activation of CD8+ and CD4+ T cells as well as CD4+Foxp3+ regulatory T cells at early time points during infection. Moreover, systemic levels of the pro-inflammatory cytokines IFN-γ and TNF-α were decreased in P. yoelii-infected mice deficient for CD11chigh DCs compared to infected RosaiDTR controls. To further elucidate the importance of CD11chigh DCs during the later phase of infection, we treated RosaiDTR/CD11c-cre and control mice with DT only from day 4 of P. yoelii infection onward. Strikingly, this approach had no impact on the activation and IFN-γ production of CD4+ and CD8+ effector T cells. These results indicate that CD11chigh DCs play a crucial role in eliciting effector T cell responses during the initial phase, but are dispensable during ongoing infection with P. yoelii.