An immune dysfunction score for stratification of patients with acute infection based on whole-blood gene expression

Dysregulated host responses to infection can lead to organ dysfunction and sepsis, causing millions of global deaths each year. To alleviate this burden, improved prognostication and biomarkers of response are urgently needed. We investigated the use of whole-blood transcriptomics for stratification of patients with severe infection by integrating data from 3149 samples from patients with sepsis due to community-acquired pneumonia or fecal peritonitis admitted to intensive care and healthy individuals into a gene expression reference map. We used this map to derive a quantitative sepsis response signature (SRSq) score reflective of immune dysfunction and predictive of clinical outcomes, which can be estimated using a 7- or 12-gene signature. Last, we built a machine learning framework, SepstratifieR, to deploy SRSq in adult and pediatric bacterial and viral sepsis, H1N1 influenza, and COVID-19, demonstrating clinically relevant stratification across diseases and revealing some of the physiological alterations linking immune dysregulation to mortality. Our method enables early identification of individuals with dysfunctional immune profiles, bringing us closer to precision medicine in infection.peer-reviewe

Inflammatory monocytes mediate control of acute alphavirus infection in mice

<div><p>Chikungunya virus (CHIKV) and Ross River virus (RRV) are mosquito-transmitted alphaviruses that cause debilitating acute and chronic musculoskeletal disease. Monocytes are implicated in the pathogenesis of these infections; however, their specific roles are not well defined. To investigate the role of inflammatory Ly6C^hiCCR2⁺ monocytes in alphavirus pathogenesis, we used CCR2-DTR transgenic mice, enabling depletion of these cells by administration of diptheria toxin (DT). DT-treated CCR2-DTR mice displayed more severe disease following CHIKV and RRV infection and had fewer Ly6C^hi monocytes and NK cells in circulation and muscle tissue compared with DT-treated WT mice. Furthermore, depletion of CCR2⁺ or Gr1⁺ cells, but not NK cells or neutrophils alone, restored virulence and increased viral loads in mice infected with an RRV strain encoding attenuating mutations in nsP1 to levels detected in monocyte-depleted mice infected with fully virulent RRV. Disease severity and viral loads also were increased in DT-treated CCR2-DTR⁺;<i>Rag1</i>^-/- mice infected with the nsP1 mutant virus, confirming that these effects are independent of adaptive immunity. Monocytes and macrophages sorted from muscle tissue of RRV-infected mice were viral RNA positive and had elevated expression of <i>Irf7</i>, and co-culture of Ly6C^hi monocytes with RRV-infected cells resulted in induction of type I IFN gene expression in monocytes that was <i>Irf3</i>;<i>Irf7</i> and <i>Mavs</i>-dependent. Consistent with these data, viral loads of the attenuated nsP1 mutant virus were equivalent to those of WT RRV in <i>Mavs</i>^-/- mice. Finally, reconstitution of <i>Irf3</i>^<i>-/-</i>;<i>Irf7</i>^-/- mice with CCR2-DTR bone marrow rescued mice from severe infection, and this effect was reversed by depletion of CCR2⁺ cells, indicating that CCR2⁺ hematopoietic cells are capable of inducing an antiviral response. Collectively, these data suggest that MAVS-dependent production of type I IFN by monocytes is critical for control of acute alphavirus infection and that determinants in nsP1, the viral RNA capping protein, counteract this response.</p></div

Induction of type I IFN expression in monocytes requires virus infection.

<p>(A-B) Enriched WT bone marrow monocytes were co-cultured with Vero cells infected with RRV-GFP (n = 3/group). After 18 h of co-culture, GFP expression in Vero cells and monocytes was measured by flow cytometry. (A) Shown are representative histograms. (B) The percent GFP⁺ monocytes and Vero cells. Data are representative of two independent experiments. (C-D) Enriched WT bone marrow monocytes were co-cultured with uninfected or RRV-GFP-infected Vero cells in the presence or absence of 1 μM Latrunculin B. After 18 h of co-culture, (C) IFNα2 mRNA expression level in monocytes was quantified by qRT-PCR. Data are normalized to 18S rRNA levels and are expressed as the relative expression (<i>n</i>-fold increase) over expression in uninfected Vero cells without monocytes. (D) The percent GFP⁺ Vero cells and monocytes were measured by flow cytometry based on GFP expression within the CD45^- Vero cells and the CD45⁺ monocytes. Data are combined from two independent experiments. <i>P</i> values were determined by one-way ANOVA with a Tukey’s multiple comparison test.</p

MAVS-dependent induction of type I IFN in monocytes during RRV infection.

<p>(A) IFNα2 mRNA expression levels in enriched WT bone marrow monocytes co-cultured for 18 h with uninfected, RRV-T48- or RRV-T48-nsP1^6M-infected-infected Vero cells (n = 6/group). (B) IFNα2 mRNA expression levels in enriched WT or <i>Irf3</i>^-/-;<i>Irf7</i>^-/- bone marrow monocytes co-cultured for 18 h with uninfected or RRV-T48-infected-infected Vero cells (n = 6/group). (C) IFNα2 mRNA expression levels in enriched WT or <i>Mavs</i>^-/- bone marrow monocytes co-cultured for 18 h with uninfected or RRV-T48-infected-infected Vero cells (n = 6-9/group). Data are normalized to 18S rRNA levels and are expressed as the relative expression (<i>n</i>-fold increase) over expression in uninfected Vero cells without monocytes. Data are combined from at least two independent experiments. <i>P</i> values were determined by one-way ANOVA with a Tukey’s multiple comparison test. *, <i>P</i> <0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001.</p

Depletion of CCR2⁺ cells enhanced viral loads in tissues and the severity of disease during acute CHIKV infection.

<p>WT (n = 9) or CCR2-DTR (n = 6) C57BL/6 mice were inoculated in the left rear footpad with 1,000 PFU of CHIKV. At days -1 and +2 relative to infection, mice were i.p. administered DT. (A) The percent starting body was determined daily. At 5 dpi, (B) viral RNA levels in the contralateral ankle were quantified by qRT-PCR and (C) infectious virus in the serum was quantified by plaque assay. Data are pooled from three independent experiments. <i>P</i> values were determined by a repeated measures two-way ANOVA with a Bonferroni’s multiple comparison test (A) and by Student’s t-test (B, C). *, <i>P</i> <0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001.</p