# Reassessing Legionella pneumophila Recognition during Intracellular Infection of Human Macrophages

> **NIH NIH R21** · NORTHWESTERN UNIVERSITY · 2020 · $187,400

## Abstract

PROJECT SUMMARY
Legionella pneumophila (Lp) causes Legionnaires' disease. Upon inhalation, Lp infects macrophages in the
lung, and this is followed by severe inflammation. During infection of macrophages, Lp triggers the secretion of
pro-inflammatory cytokines. Based largely upon studies done in murine models, the immune recognition of Lp
is triggered by certain surface and endosomal Toll-like receptors (TLRs), cytosolic NOD-like receptors, RIG-I-
like receptors, and inflammasomes. Recently, in order to discern the effect of Lp type II protein secretion on
cytokine elicitation, we used shRNA to generate a panel of human macrophages that are deficient in individual
immune components. While utilizing these new reagents, we made a number of observations concerning the
recognition of Lp by human macrophages that deviate from / expand upon results obtained with murine cells.
These findings include i) a minimal (rather than major) role for MyD88-dependent TLR signaling, ii) a larger
role for the TRIF and TRAM adaptors and by extension endosomal TLR3 and/or endosomal TLR4, iii) a major
role for the TBK-1 adapter, cGAS and STING, and by inference cytosolic DNA-sensing, and iv) a new role for
PKR and likely other cytosolic RNA-sensors. These data call into question the field's long-standing reliance on
murine models and indicate a compelling need to further define the innate immune response in human
macrophage models. Also, some of our results echo findings from human epidemiological studies that have
been largely ignored in recent years. Therefore, we propose to utilize knockdown protocols to discern, in the
context of Lp infection of human macrophages (both U937 cell lines and those derived from peripheral blood
mononuclear cells), the relative importance of TLR3 and TLR4, cytosolic DNA sensors DDX41, DNA-PK, IFI16,
and MRE11, and cytosolic RNA sensors DDX3, DHX9, DHX33, DDX1/DDX21/DHX36. In order to ensure an
unbiased investigation of Lp recognition by human macrophages, we will also examine knockdowns of C-type
lectin receptors (Mcl, Mincle, Dectin-1, Dectin-2), which have heretofore been entirely absent from Lp studies.
Examination of a parallel set of knockdowns in murine macrophages will allow us to unequivocally draw the
distinction between human vs. murine infection. This line of inquiry should more readily translate into an
increased understanding of and potential treatment for Lp-mediated disease in humans. Also, findings
obtained with Lp have implications for other bacteria, especially intracellular pathogens of human
macrophages; e.g., TLR3 and the other nucleic acid-sensing pathways have been largely studied in viral
infections, and lectin receptors have been mainly examined with fungi.

## Key facts

- **NIH application ID:** 9823850
- **Project number:** 5R21AI137458-02
- **Recipient organization:** NORTHWESTERN UNIVERSITY
- **Principal Investigator:** NICHOLAS P CIANCIOTTO
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $187,400
- **Award type:** 5
- **Project period:** 2018-11-15 → 2021-10-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9823850

## Citation

> US National Institutes of Health, RePORTER application 9823850, Reassessing Legionella pneumophila Recognition during Intracellular Infection of Human Macrophages (5R21AI137458-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9823850. Licensed CC0.

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