Francisella tularensis
Live Vaccine Strain training of murine alveolar and bone marrow-derived macrophages
Authors: Khan Hamda , Bhargava Varunika , Elkins Karen L. 
Published: June 2024

Traditionally, successful vaccines rely on specific adaptive immunity by activating lymphocytes with an attenuated pathogen, or pathogen subunit, to elicit heightened responses upon subsequent exposures. However, recent work with
Mycobacterium tuberculosis
and other pathogens has identified a role for “trained” monocytes in protection through memory-like but non-specific immunity. Here, we used an
in vitro
co-culture approach to study the potential role of trained macrophages, including lung alveolar macrophages, in immune responses to the Live Vaccine Strain (LVS) of
Francisella tularensis. F. tularensis
is an intracellular bacterium that replicates within mammalian macrophages and causes respiratory as well as systemic disease. We vaccinated mice with
F. tularensis
LVS and then obtained lung alveolar macrophages, or derived macrophages from bone marrow. LVS infected and replicated comparably in both types of macrophages, whether naïve or from LVS-vaccinated mice. LVS-infected macrophages were then co-cultured with either naïve splenocytes, splenocytes from mice vaccinated intradermally, or splenocytes from mice vaccinated intravenously. For the first time, we show that immune (but not naïve) splenocytes controlled bacterial replication within alveolar macrophages, similar to previous results using bone marrow-derived macrophage. However, no differences in control of intramacrophage bacterial replication were found between co-cultures with naïve macrophages or macrophages from LVS-vaccinated mice; furthermore, nitric oxide levels and interferon-gamma production in supernatants were largely comparable across all conditions. Thus, in the context of
in vitro
co-cultures, the data do not support development of trained macrophages in bone marrow or lungs of mice vaccinated with LVS intradermally or intravenously.

IMPORTANCE

The discovery of non-specific “trained immunity” in monocytes has generated substantial excitement. However, to date, training has been studied with relatively few microbes (e.g.,
Mycobacterium bovis
Bacille Calmette-Guérin, a live attenuated intracellular bacterium used as a vaccine) and microbial substances (e.g., LPS), and it remains unclear whether training during infection is common. We previously demonstrated that vaccination of mice with
Francisella tularensis
Live Vaccine Strain (LVS), another live attenuated intracellular bacterium, protected against challenge with the unrelated bacterium
Listeria monocytogenes
. The present study therefore tested whether LVS vaccination engenders trained macrophages that contributed to this protection. To do so, we used a previous
in vitro
co-culture approach with murine bone marrow-derived macrophages to expand and study lung alveolar macrophages. We demonstrated that alveolar macrophages can be productively infected and employed to characterize interactions with LVS-immune lymphocytes. However, we find no evidence that either bone marrow-derived or alveolar macrophages are trained by LVS vaccination.