Distinct Innate Responses are Induced by Attenuated Salmonella enterica serovar Typhimurium Mutants

Daniel A Powell; Lydia M Roberts; Hannah E Ledvina; Gregory D Sempowski; Roy Curtiss, III; Jeffrey A Frelinger

doi:10.1016/j.cellimm.2015.10.002

. Author manuscript; available in PMC: 2017 Jan 1.

Published in final edited form as: Cell Immunol. 2015 Oct 22;299:42–49. doi: 10.1016/j.cellimm.2015.10.002

Distinct Innate Responses are Induced by Attenuated Salmonella enterica serovar Typhimurium Mutants

Daniel A Powell ^a,^*, Lydia M Roberts ^a,^d, Hannah E Ledvina ^a,^e, Gregory D Sempowski ^b, Roy Curtiss III ^c,^f, Jeffrey A Frelinger ^a

PMCID: PMC4704447 NIHMSID: NIHMS736425 PMID: 26546408

Abstract

Upon bacterial infection the host cells generate a wide variety of cytokines. Genetic attenuation of bacterial physiological pathogens can be accomplished not only by disruption of normal bacterial processes, but also by the loss of the ability to redirect the host immune system. We examined nine attenuated Salmonella Typhimurium mutants for their ability to replicate as well as the cytokines produced after infection of Bone Marrow Derived Macrophages (BMDM). Infection of BMDM with attenuated Salmonella mutants led to host cytokine patterns distinct from those that followed WT infection. Surprisingly, each bacterial mutant had a unique cytokine signature. Because some of the mutants induced an IL-10 response not seen in WT, we examined the role of IL-10 on Salmonella replication. Surprisingly, addition of IL-10 before or concurrent with infection restricted growth of WT Salmonella in BMDM. Bacterial attenuation is not a single process and results in attenuated host responses, which result in unique patterns for each attenuated mutants.

Introduction

Salmonella enterica serovar Typhimurium is a Gram-negative rod shaped bacterium that causes severe gastroenteritis. Salmonella infections impose a large global health burden with approximately 200 million to 1 billion cases annually [1]. Salmonella exposure poses a significant public health threat and can come from a wide variety of sources including raw eggs, raw meat, and pet feces.

The infectious cycle of Salmonella has been well-characterized in mice as well as human cell lines [2-4]. Salmonella initially enters the cell using genes encoded on Salmonella Pathogenicity Island-I (SPI-1). Proteins encoded by SPI-1 are recognized by host cells and cellular responses lead to the recruitment of PMNs and activation of host NFκB [5-8]. Salmonella is phagocytosed by the host mucosal macrophages (M cells). The bacteria resides in an acidic membrane enclosed vacuole, the Salmonella Containing Vacuole (SCV). In response to the acidic environment Salmonella upregulates genes on the second pathogenicity island (SPI-2) which allow the bacterium to exit the SCV to the cytosol where it replicates [9-13]. Counterintuitively, engagement of host TLRs are critical in upregulation of SPI-2 genes and bacterial replication [14].

While the virulence factors carried on these two pathogenicity islands have been well-characterized, we sought to discover other Salmonella encoded genes that are able to actively reshape the host immune responses. This differs from simple immune avoidance mechanisms used by pathogens to avoid destruction such as Streptococcus pneumoniae masking antibody targets of the host response by creating a thick polysaccharide coat or Neisseria under going phase variation. Here we focus on specific host immune responses that are actively stimulated or blocked by various attenuated Salmonella Typhimurium mutants. Attenuation of bacterial pathogens leads to a unique a varied cytokine profiles, not simply more cytokine than wild type infection.

Materials and Methods

Bacterial Strains and Growth Conditions

The strains of Salmonella Typhimurium used in this study and their sources are listed in Table 1. ΔpabA/B, ΔhisG/crp, ΔphoP/hisG/rpsL, ΔsifA, and ΔrecA are standard genetic deletions. The phoP/Q^-, fur^-, crp^-, and recA^- were designed as delayed attenuation mutants where the native promoter has been replaced with an arabinose inducible promoter [15-19]. All strains were grown in either Luria Broth (LB) or Purple Broth (PB) (Sigma-Aldrich) with arabinose as appropriate. Log phase Salmonella bacterial cultures were used to infect cell lines. 1mL of log phase culture was measured for OD₆₀₀ and compared to empirically determined standards for each strain. Final inoculation counts were determined by serial dilution and plating on LB agar. For the arabinose controlled promoters Salmonella and target cells were grown in the presence or absence of 0.2% arabinose to control gene expression.

Table 1. Strains used in this study.

Strain Number	Description	Function	Source	Strain Designation
χ3761	WT Salmonella	-	UK-1	WT
χ 8442	ΔpabA1516 ΔpabB232	Aminobenzoate auxotroph	UK-1	ΔpabA/B
χ 8499	ΔhisG Δcrp28	Histidine synthesis, sugar transport	SL-1344	ΔhisG/crp
χ 8770	ΔphoP233 ΔhisG ΔrpsL	Two-component kinase, histidine synthesis, Strep resistance	SL-1344	ΔphoP/hisG/rpsL
χ 8926	ΔsifA26	SPI-2 secreted effector	UK-1	ΔsifA
χ 9199	ΔP_phoPQ107∷TTaraCP_BADphoPQ ΔaraBAD23	Two-component kinase and sensor	UK-1	phoP/Q^-
χ 9201	ΔP_fur33∷TTaraCP_BADfur ΔaraBAD23	Ferric uptake regulator	UK-1	fur^-
χ 9202	ΔP_crp527∷TTaraCP_BADcrp ΔaraBAD23	Sugar transport	UK-1	crp^-
χ 9833	ΔrecA62	Homologous recombination/DNA repair	UK-1	ΔrecA
χ 11144	ΔP_recA74∷TTaraCP_BADrecAΔaraBAD23	Homologous recombination/DNA repair	UK-1	recA^-

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Mice

C57BL/6J (B6), C57BL/10ScNJ (TLR4^-/-), and B6.129P2-Il10^tm1Cgn/J (IL-10^-/-) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and then bred in-house. All mice were housed under specific-pathogen-free conditions at the University of Arizona and used in accordance with the Institutional Animal Care and Use Committees.

Cell Lines and Bone Marrow Derived Macrophage Production

J774.1 cells were obtained from ATCC (Manassas, VA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Atlas), L-glutamine (HyClone), sodium pyruvate (HyClone), and penicillin-streptomycin (Life Technologies). Medium was replaced with antibiotic-free medium 24 h prior to inoculation with Salmonella. For production of primary macrophages bone marrow cells were flushed from mouse femurs and incubated for seven days on non-tissue culture-treated 15-cm² dishes with L cell-conditioned medium containing GM-CSF. Following differentiation, non-adherent cells were removed by multiple washes with PBS and bone marrow-derived macrophages were removed from plates by scraping. Cells were then washed and resuspended in Complete DMEM without antibiotics for infection.

In vitro growth assays

A total of 2 × 10⁵ cells/well (BMDM or J774) were seeded into a 96-well plate for Salmonella intracellular growth assays and incubated two hours to allow adherence to the plate. In some experiments recombinant murine interlukin-10 (Peprotech) (rIL-10) was added either two hours before or at the time of infection. Cells were inoculated at a range (100-3.125) of multiplicities of infection (MOI) the ratio of bacteria to eucaryotic cells. Infection was facilitated by centrifugation at 300 × g for 5 min. Cells were incubated for one hour with bacteria, and the medium was then removed. Fresh medium containing 50 μg/mL gentamicin (Sigma) was added to kill extracellular bacteria. One hour after gentamicin addition, medium was removed, and the cells were washed twice before the addition of fresh antibiotic free medium. All culture medium used in the arabinose inducible promoter system experiments contain 0.2% arabinose as indicated in the figures. To determine intracellular growth, medium was removed at indicated time points post-infection, and 1 mL of PBS was added to the cells. Cells were removed from the plate by vigorous pipetting. Cells were lysed by vortexing at maximal speed for one minute. Serial 1:10 dilutions of the lysate were made and plated onto LB agar. The resulting colonies were counted 24 hours later.

Luminex Analysis

Cytokines/chemokines in culture supernatant were measured using a 20-analyte Luminex bead-based approach according to the manufacturer's protocol (Invitrogen), using a BioPlex 100/200 array reader (Bio-Rad Laboratories). Using integrated cytokine/chemokine standard curves, the assay reports the following mouse analytes in pg/mL: fibroblast growth factor (FGF) basic, granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p40/p70), IL-13, IL-17, IP-10, KC, monocyte chemoattractant protein 1 (MCP-1), monokine induced by IFN-γ (MIG), macrophage inflammatory protein 1α (MIP-1α), tumor necrosis factor alpha (TNF-α), and vascular endothelial growth factor. A five-parameter nonlinear logistic regression model was used to establish a standard curve and to estimate the probability of occurrence of a concentration at a given point Upper and lower levels of quantification were determined by use of BioPlex Manager software based on goodness of fit and percent recovery. Calculated pg/mL for experimental specimens were multiplied by the inherent assay dilution factor (df = 2) and reported as final observed pg/mL.

Statistical Analysis

Statistical analysis was carried out using Graph Pad Prism Version 6 (La Jolla, CA). The statistical test used for each experiment is described in the figure legends.

Results

Kinetics of Salmonella Invasion and Replication

We chose the Salmonella mutants were chosen for this study because they were all attenuated in mouse infection models and imparted varying levels of protective immunity against later lethal challenge, however their behavior in cells has been poorly characterized [15-19]. First we sought to determine if each mutant was able to invade and replicate in Bone Marrow Derived Macrophages (BMDM), as described above. At two and six hours post infection the cells were lysed and bacterial burdens were determined by serial dilution and plating. Interestingly, 8/9 mutants had significantly lower counts at two hours indicating that these strains are deficient in invasion as compared to the WT strain (Figure 1). The one strain that showed similar invasion to WT was the ΔhisG/rpsL/phoP mutant. We then compared the growth of each strain from two to six hours. Two strains, the ΔhisG/crp mutant and the phoP/Q^- mutant failed to grow. All other mutant strains showed 2-3 fold growth, while the WT strain showed about 5 fold growth, indicating that all of the mutants grew more slowly in BMDM then WT.

C57BL/6 BMDM were infected with the indicated *Salmonella* strain at a MOI of 5. Wells were lysed at 2 (open bars) and 6 (filled bars) and enumerated by serial dilution. Values are represented as mean and SEM for 6 wells per condition. Data is from one representative of 3 experiments. All mutant strains are significantly different from WT in growth (P<.001) Stars indicate ****, p<0.0001, ***, p<0.001, **, p<0.01, bars show the significance of growth of individual strain between 2 and 6 hours. Student's t test corrected for multiple comparisons.

Host Cytokine Patterns Induced Upon Salmonella Infection Are Strain Specific

We determined if the host innate immune system was differentially activated by these mutant Salmonella strains as opposed to simply inducing more of the same cytokines. We infected BMDM in triplicate with WT or mutant Salmonella at a range of different MOI. Salmonella were allowed to invade host BMDM as above. At six hours post infection the supernatant was harvested and frozen at -80°C until analyzed for cytokine/chemokine levels. A broad panel of cytokines/chemokines was chosen to allow for unbiased examination of host response.

Of the 20 cytokines tested 11 (FGF-Basic, GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-13, IL-17, KC, MIG and VEGF) were not detected at any MOI and are not shown. Importantly, each mutant shows a unique pattern of cytokine expression (Table 2). IL-1α was only observed in WT infection at the highest MOI of 100, it was not detected upon infection with most of the attenuated mutant strains, although two strains (phoP/Q^- and fur^-) showed some IL-1α in one replicate at the highest MOI (Figure S1). It is not clear if this is produced by new synthesis or increased processing of existing IL-1α precursors.

Table 2. Fold change in cytokines compared to WT Salmonella infection in WT BMDM.

MOI 100 WT BMDM	IL-1α	IL-1β	IL-6	MCP-1	TNF-α	MIP-1α	IL-10	IL-12p40/70	MOI 50 TLR4-/- BMDM	IL-1β	IL-6	MCP-1	TNF-α	MIP-1α	IL-10	IL-12p40/70
χ8442 ΔpabA/B									χ8442 ΔpabA/B
χ8499 ΔhisG/crp									χ8499 ΔhisG/crp
χ8770 ΔphoP/hisG/rpsL									χ8770 ΔphoP/hisG/rpsL
χ8926 ΔsifA									χ8926 ΔsifA
χ9199 phoP/Q^-									χ9199 phoP/Q^-
χ9201 fur^-									χ9201 fur^-
χ9202 crp^-									χ9202 crp^-
χ9833 ΔrecA									χ9833 ΔrecA
χ11144 recA^-									χ11144 recA^-
MOI 25 WT BMDM	IL-1α	IL-1β	IL-6	MCP-1	TNF-α	MIP-1α	IL-10	IL-12p40/70	MOI 12.5 TLR4-/- BMDM	IL-1β	IL-6	MCP-1	TNF-α	MIP-1α	IL-10	IL-12p40/70
χ8442 ΔpabA/B									χ8442 ΔpabA/B
χ8499 ΔhisG/crp									χ8499 ΔhisG/crp
χ8770 ΔphoP/hisG/rpsL									χ8770 ΔphoP/hisG/rpsL
χ8926 ΔsifA									χ8926 ΔsifA
χ9199 phoP/Q^-									χ9199 phoP/Q^-
χ9201 fur^-									χ9201 fur^-
χ9202 crp^-									χ9202 crp^-
χ9833 ΔrecA									χ9833 ΔrecA
χ11144 recA^-									χ11144 recA^-
MOI 6.25 WT BMDM	IL-1α	IL-1β	IL-6	MCP-1	TNF-α	MIP-1α	IL-10	IL-12p40/70	MOI 3.125 TLR4-/- BMDM	IL-1β	IL-6	MCP-1	TNF-α	MIP-1α	IL-10	IL-12p40/70
χ8442 ΔpabA/B									χ8442 ΔpabA/B
χ8499 ΔhisG/crp									χ8499 ΔhisG/crp
χ8770 ΔphoP/hisG/rpsL									χ8770 ΔphoP/hisG/rpsL
χ8926 ΔsifA									χ8926 ΔsifA
χ9199 phoP/Q^-									χ9199 phoP/Q^-
χ9201 fur^-									χ9201 fur^-
χ9202 crp^-									χ9202 crp^-
χ9833 ΔrecA									χ9833 ΔrecA
χ11144 recA^-									χ11144 recA^-
Fold Change to WT
<0.75
2.00-4.99
5.00-9.99
>10.00

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In contrast, IL-1β was found only after infection with the phoP/Q^- and ΔrecA mutant at the intermediate MOIs, 25-6.25 and only in some of the triplicate wells. IL-1β was also detected following infection with the fur^- mutant infection at MOIs of 100 and 12.5. Interestingly, the recA^- mutant showed no detectable IL-1β at any MOI indicating that the mechanism of the bacterial mutation (deletion vs promoter swap) alters the host response (Figure S2). The inflammatory cytokine IL-6 showed an inverse dose-dependent response in the WT strain. After WT infection, no cytokine was detected at the highest MOIs tested 100, 50, 25 whereas robust IL-6 production was detected at the lower MOIs. IL-6 was not detected at any MOI upon infection with the ΔpabAB, ΔhisG/crp, or ΔphoP/rpsL/hisG mutants. The remaining mutants showed an increased production of IL-6 at the higher MOIs as compared to WT infection. At the lower MOIs WT infection led to more IL-6 production than any of the mutants (Figure S3). Among other cytokines showing an inverse dose-dependent response are MCP-1 (Figure S4) and TNF-α (Figure S5). They are higher in all of the mutants than WT at the higher MOIs at lower MOIs the WT is higher of equal to all of the mutant strains. MIP-1α showed a similar dose dependence in all of the mutants, except the recA^- mutant that induced higher levels at most MOIs tested (Figure S6). One other cytokine showing an interesting pattern among mutants was Interferon-γ Induced Protein 10 (IP-10). The mutant Salmonella infections showed a variable production of IP-10 as compared to WT. Infection with phoP/Q^- and fur^- mutants showed relatively similar levels to WT infection. The crp-, ΔpabA/B, ΔhisG/crp, ΔphoP/hisG/rpsL, and ΔsifA mutants induced slightly higher IP-10 than WT at the higher MOI and but lagged behind WT at the lower MOI. Again with the recA mutants the mechanism of attenuation effects the cytokine pattern. The arabinose promoter recA^- strain in arabinose free medium induced IP-10 at lower levels than WT at lower MOIs, and higher levels than WT at higher MOIs. The gene deletion strain ΔrecA consistently induced IP-10 at higher or equal levels than WT at all MOIs tested (Figure S7).

Two cytokines, IL-10 and IL-12p70, showed consistent changes in some mutant strains as compared to WT. IL-10 was not detected upon WT infection at any MOI. In contrast, infection with the phoP/Q^- mutant and the recA^- mutants induced IL-10 production at most of the MOIs tested (Figure S8). IL-10 was also detected at some MOIs in ΔphoP/hisG/crp stain. IL-12p70 was detected after infection with most mutant Salmonella strains, the exceptions were the ΔpabA/B and ΔhisG/crp strains that showed no detectable levels of IL-12p70 at any MOI tested (Figure S9).

Salmonella is primarily a gastrointestinal pathogen. Since TLR4 is expressed at very low levels in the GI tract we repeated these cytokine experiments in TLR4^-/- BMDM to mimic these low TLR conditions in the gut [20-22]. Similar to infection of WT BMDM, FGF Basic, GM-CSF, IL-2, IL-5, and IL-17 were not detected at any MOI following infection with any of the strains tested. The inflammatory cytokines IL-6 and MCP-1 are completely dependent on TLR4. IL-1α showed a similar profile to the WT BMDM infections where it is only found after infection with the highest doses of WT Salmonella (Figure S10) suggesting TLR4 is not required. Unlike in WT BMDM where IL-1β was only detected in a few mutants and MOIs, most mutants induced high levels of IL-1β at every MOI tested (Figure S11). Similar “TLR4^-/- dependent” responses were seen in IL-4 (Figure S12), IL-13 (Figure S13), KC (Figure S14) and VEGF (Figure S15). These cytokines that are only seen in the absence of TLR4 highlight a need to explore inapparent innate responses. MIP-1α was detected in all the infections, but all of the mutants showed lower levels compared to WT (Figure S16). IL-10 (Figure S17) and IL-12p70 (Figure S18) were detected after infection with most of the mutant Salmonella strains, although it is important to note the uninfected production of IL-10 was higher in the TLR4^-/- BMDM.

IL-10 rescues phoPQ mutant Salmonella in-vitro growth

IL-10 is often considered to be an anti-inflammatory cytokine. We were surprised to see it upregulated in attenuated mutant infections. IL-10 was detected after infection with the phoP/Q^- and recA^- mutants at all MOIs tested. Host inflammation plays a complex role in Salmonella growth and pathogenesis. Hosts that are deficient for IL-12 or IFN-γ are particularly sensitive to Salmonella infection so it might be thought that IL-10 production would mimic that seen in hosts deficient for these inflammatory cytokines. The phoP/Q^- mutant, which induced the highest levels of IL-10 also fails to replicate in host macrophages suggesting a possible protective role for host IL-10. With this in mind we sought to determine if the phoP/Q^- mutant proliferation could be rescued in IL-10^-/-BMDM. To this end WT or IL-10^-/- BMDM were infected with either WT or phoP/Q^- Salmonella at an MOI of 5, additionally the ΔhisG/crp mutant that fails to grow in BMDM, but also fails to induce host IL-10 was included. Two and six hours post infection BMDMs were lysed and bacterial levels were determined by serial dilution and plating. The WT Salmonella showed similar invasion and replication numbers regardless of the BMDM type infected (Figure 2). The phoP/Q^- replication was rescued in the absence of host IL-10. There was no difference in invasion of either WT or IL-10^-/- BMDM. The ΔhisG/crp mutant failed to grow in either BMDM line indicating that its attenuation was independent of host IL-10 production, consistent with the lack of IL-10 detected by Luminex-based cytokine profiling.

C57BL/6 BMDM were infected with the WT (A) or Δ*phoP/Q Salmonella* at a MOI of 5. rIL-10 was added at the indicated concentration either 3 hours before (Pre) or at the time of infection. Wells were lysed at 2 (open bars) and 6 (filled bars) and enumerated by serial dilution. Values are represented as mean and SEM for 3 wells per condition. Data is from one representative of 2 experiments. Student's t test corrected for multiple comparisons.

IL-10 Restricts WT Salmonella in-vitro growth

To determine if IL-10 can restrict WT Salmonella, BMDM were treated with a range of concentrations of rIL-10 either four hours before or at the time of infection with either WT or phoP/Q^- Salmonella. Salmonella infection was performed as above and bacteria harvested two and six hours post infection. As seen previously the phoP/Q^- mutant showed no significant growth regardless of the amount of IL-10 added. Interestingly, the addition of IL-10 either before or at the time of infection restricted the growth of WT Salmonella as compared to infections in the absence of IL-10 (p<0.001) (Figure 3), indicating a host protective role for IL-10 in the infection of BMDM.

C57BL/6 (WT) or IL-10^-/- BMDM were infected with the indicated *Salmonella* strain at a MOI of 5. Wells were lysed at 2 (open bars) and 6 (filled bars) and enumerated by serial dilution. Values are represented as mean and SEM for 3 wells per condition. Data is from one representative of 2 experiments. ****, p<0.0001, ***, p<0.001, **, p<0.01 relative to 2 hour timepoint. (Student's unpaired t test)

Discussion

Comparing the host response in various mutants provide important insights into host responses to attenuated pathogens. Much of vaccine development is predicated on the assumption that attenuated organisms produce an immune response equivalent to the pathogen. Here we show that the attenuated mutants have distinct cytokine signatures. The finding that these presumptive vaccine candidates differ not only in the amount but types of cytokine induced is critical information. All of these mutants fail to cause disease in mice [8, 15-19, 23-26]. When carrying out in vivo infections there is little discernable difference amongst the mutant strains. Examining the host immune pathways activated by individual attenuated mutants highlights that the host responds to each of the attenuated mutants with a unique cytokine signature. None of the nine mutants had identical production of cytokines compared to WT Salmonella infection or one another (Table 2). The same is true in the TLR4^-/- BMDM (Table 3). Additionally, the mechanism of genetic manipulation of the bacteria also had an effect on the cytokines produced. The genetic deletion ΔrecA gave a different cytokine profile than an arabinose promoter recA^- strain in the absence of arabinose. Thus the idea that attenuated mutants produce a similar response as WT only lower in magnitude is incorrect.

Table 3. Fold change in cytokines compared to WT Salmonella infection in TLR4^-/- BMDM.

MOI 100 TLR4-/- BMDM	IL-1α	IL-1β	IL-4	IL-13	KC	VEGF	MIP-1α	IL-10	IL-12p40/70	MOI 50 TLR4-/- BMDM	IL-1α	IL-1β	IL-4	IL-13	KC	VEGF	MIP-1α
χ8442 ΔpabA/B										χ8442 ΔpabA/B
χ8499 ΔhisG/crp										χ8499 ΔhisG/crp
χ8770 ΔphoP/hisG/rpsL										χ8770 ΔphoP/hisG/rpsL
χ8926 ΔsifA										χ8926 ΔsifA
χ9199 phoP/Q^-										χ9199 phoP/Q^-
χ9201 fur^-										χ9201 fur^-
χ9202 crp^-										χ9202 crp^-
χ9833 ΔrecA										χ9833 ΔrecA
χ11144 recA^-										χ11144 recA^-
MOI 25 TLR4-/- BMDM	IL-1α	IL-1β	IL-4	IL-13	KC	VEGF	MIP-1α	IL-10	IL-12p40/70	MOI 12.5 TLR4-/- BMDM	IL-1α	IL-1β	IL-4	IL-13	KC	VEGF	MIP-1α
χ8442 ΔpabA/B										χ8442 ΔpabA/B
χ8499 ΔhisG/crp										χ8499 ΔhisG/crp
χ8770 ΔphoP/hisG/rpsL										χ8770 ΔphoP/hisG/rpsL
χ8926 ΔsifA										χ8926 ΔsifA
χ9199 phoP/Q^-										χ9199 phoP/Q^-
χ9201 fur^-										χ9201 fur^-
χ9202 crp^-										χ9202 crp^-
χ9833 ΔrecA										χ9833 ΔrecA
χ11144 recA^-										χ11144 recA^-
MOI 6.25 TLR4-/- BMDM	IL-1α	IL-1β	IL-4	IL-13	KC	VEGF	MIP-1α	IL-10	IL-12p40/70	MOI 3.125 TLR4-/- BMDM	IL-1α	IL-1β	IL-4	IL-13	KC	VEGF	MIP-1α
χ8442 ΔpabA/B										χ8442 ΔpabA/B
χ8499 ΔhisG/crp										χ8499 ΔhisG/crp
χ8770 ΔphoP/hisG/rpsL										χ8770 ΔphoP/hisG/rpsL
χ8926 ΔsifA										χ8926 ΔsifA
χ9199 phoP/Q^-										χ9199 phoP/Q^-
χ9201 fur^-										χ9201 fur^-
χ9202 crp^-										χ9202 crp^-
χ9833 ΔrecA										χ9833 ΔrecA
χ11144 recA^-										χ11144 recA^-
Fold Change to WT
<0.75
2.00-4.99
5.00-9.99
>10.00

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Our experiments showed very little IL-1α being produced by BMDM after Salmonella infection, protein was only detected in WT infection at the highest MOI (100) tested. IL-1α has been previously shown to be produced by human monocytes upon incubation with high levels (∼1ug/mL) of porin protein from Salmonella [27, 28]. This dependency on high levels of porin coupled with the WT Salmonella invading at a higher efficiency than the mutants, and thus having more porin per target cell, may explain why no other conditions resulted in IL-1α production.

One surprising result was the lack of IL-1β detected in our assay system. Other groups have previously shown Salmonella induced high levels of IL-1β [29, 30]. This IL-1β production has been shown to be important for both Salmonella pathogenesis and redirection of the host immune system. Perkins et al. showed high levels of transcript at four and six hours post infection in peritoneal macrophages, whereas Franchi et al. showed moderate levels of protein at 16 hours from BMDM, but much higher levels from intestinal mononuclear phagocytes (iMPs). Perhaps the IL-1β mRNA in our system is not translated six hours post infection or the protein is produced at low levels and signaling in an autocrine manner and so consumed. Testing of alternate time points as well as RT-PCR can be carried out in the future. Of note, while we did detect IL-1β in the WT infection of TLR4^-/- BMDM, almost every mutant induced detectable levels of IL-1β, suggesting that in this system TLR4 signaling can suppress IL-1β.

Three cytokines showed a complete dependence of TLR4, IL-6, TNF-α and MCP-1. These proinflammatory cytokines, hallmarks of LPS stimulation of TLR4, were absent in the TLR4^-/- infections. Interestingly the mutants tested showed similar dose-dependent responses as compared to WT infection. At higher MOIs most of the mutants showed increased cytokine, while at lower MOIs the WT Salmonella induced more IL-6, TNF-α and MCP-1. This was obviously dependent on LPS structure. The only mutants in the tested panel that have a direct effect on lipid A structure are the phoP/Q^- and ΔhisG/crp/phoP mutants. In response to low pH, Fe⁺³, Mg⁺² or oxygen the two component system phoPQ regulates genes in the lipid A modification pathway, namely the pagL and pagP (Pho Activated Genes) a deacylase and acylase respectively, and the pmrAB two component system which controls addition of ethanolamine and aminoarabinose to the terminal phosphates [31-33]. Under normal media growth conditions in LB or Purple Broth the PhoPQ system is inactive, thus the lipid A structure of all the strains tested should be identical. All mutants strains having the same lipid A structure but many inducing much higher levels of TLR4 dependent cytokines at the higher MOIs indicate a possible downregulation of TLR4 induced inflammation by WT Salmonella.

MIP-1α secretion by Peyer's Patch cells has been shown to lead to immune damage. Blocking of MIP-1α secretion leads to increased CD4⁺ CD25⁺ Regulatory T Cells and decreased immunopathology [34]. In WT BMDM model we once again see a dose-dependent production of MIP-1α in the mutants where at high MOI most mutant infections show increased MIP-1α secretion compared to WT but at lower MOI we see the reverse. This seems to fit with the TLR4 dependent pattern we see in the MCP-1 and IL-6. MIP-1α is generally driven by LPS stimulation of TLR4. Unlike IL-6 and MCP-1 we see MIP-1α secretion in the TLR4^-/- BMDM infection model, indicating that there is a TLR4 independent pathway that also induces production of MIP-1α. Also of note, in the TLR4^-/-BMDM infection almost all of the mutants showed lowered MIP-1α production at all but the highest MOI. Recent work has indicated the B subunits of a AB5 toxin ArtAB (ADP- ribosylating toxin) in Salmonella triggering host production of MIP-1α [35]. The decreased bacterial load and replication may explain the lowered MIP-1α production in the TLR4^-/- BMDM.

Humans deficient for IL-12R are extremely sensitive to Salmonella infections, indicating an important role for IL-12 in host defense against Salmonella [36, 37]. Most of our mutants showed an increase in IL-12 production compared to WT infection. Of note, the metabolism mutants, ΔpabA/B and ΔhisG/crp, failed to induce greater IL-12 production. These mutants have a much lower cytokine profile compared to the other mutants.

Some cytokines (IL-4, IL-13, KC and VEGF) were only induced in the absence of TLR4, suggesting an inhibitory effect of TLR signaling. All of these cytokines are signatures of a Th2 like response. Inflammatory signals through TLR4 potentiate Th1 responses. In the absence of TLR4, it makes sense that we could see a stronger Th2 response. However none of Th2 skewing cytokines are produced in the WT Salmonella infection of TLR4^-/- BMDM. This lack of production in WT infection indicates it is not simply a lack of TLR4 that allows for IL-4, IL-13, KC and VEGF, but particular bacterial phenotypes in the absence of TLR4.

Addition of arabinose to the arabinose inducible promoter strains rescued IL-10, IL-12, IL-12p40/70 and VEGF production. Some cytokine production from mutant Salmonella infection was not rescued by addition of arabinose. In the TLR4^-/- BMDM IL-1β, IL-4, and KC showed nearly equal levels regardless of arabinose additions. This may be due to differential regulation of the genes under the new promoter or a possible second site mutation in these strains.

Continuing experiments removing the dominant signal, like TLR4 in Salmonella or TLR2 in Francisella, allow for the discovery of possible other immune responses that are manipulated by pathogens [38]. These subdominant signals, while not likely directly involved in pathogenesis and survival, can allow insight into mechanisms of host subversion that might be applicable to other pathogens of interest.

The IL-10 restriction of Salmonella replication in BMDM was surprising. Many groups have shown data that IL-10 allows for rapid Salmonella growth and spread in animal models [39-43]. Our experiments carried out in BMDMs seem to fit with the need for Salmonella to use inflammation from the host to upregulate SPI-2 genes in order to replicate in the cytoplasm. Further work will be needed to determine if IL-10, like the loss of TLR2/4/9, can inhibit SPI-2 transcription and translation.

Overall these results support a revised model of the immune response to an attenuated pathogen. Infection with attenuated strains of similar phenotypes, like lowered invasion or slower growth, does not result in the same immune response as WT bacteria, only of a lower magnitude. Instead, it means that a loss of function mutant can result in both new cytokine responses not seen in WT infection and loss of other cytokine responses. Both of these changes give clues to how the bacteria are modifying host responses. It is easy to see how a loss of function in a bacterial gene that suppresses the immune response would result in a new cytokine secretion of one that was previously suppressed. This is the classic loss of a suppressor. It is more difficult to see how a loss of function mutant would result in the loss of a cytokine response. In our case loss of phoPQ and fur result in the loss of MIP-1α at all of the MOIs tested. The simplest explanation would be either the direct gene products (or a molecule regulated by them) results in the stimulation of MIP-1α. However that is difficult to reconcile with the observation that many different mutations result in the downregulation of the same cytokine, such as IL-1α. Altogether, our data suggests that we need to rethink the impact of attenuation on the immune response, and develop more nuanced models of how attenuation impacts immunity.

Supplementary Material

Figure S1: Detection of IL-1α in Culture Supernatants of WT BMDM

C57Bl/6 BMDM were infected with the indicated MOI of either WT or mutant Salmonella. For arabinose positive conditions, both BMDM and Salmonella were grown in 0.2% arabinose for the entire experiment, including overnight and subculture of Salmonella. 6 hours after infection the supernatants were removed and analyzed by Luminex. Infections were carried out in triplicate. The solid line indicates the limit of detection for the assay. Mutants that induced no detectable cytokine are not included.

Figure S2: Detection of IL-1β in Culture Supernatants of WT BMDM

Figure S3: Detection of IL-6 in Culture Supernatants of WT BMDM

Figure S4: Detection of MCP-1 in Culture Supernatants of WT BMDM

Figure S5: Detection of TNF-α in Culture Supernatants of WT BMDM

Figure S6: Detection of MIP-1α in Culture Supernatants of WT BMDM

Figure S7: Detection of IP-10 in Culture Supernatants of WT BMDM

C57Bl/6 BMDM were infected with the indicated MOI of either WT or mutant Salmonella. For arabinose positive conditions, both BMDM and Salmonella were grown in 0.2% arabinose for the entire experiment, including overnight and subculture of Salmonella. 6 hours after infection the supernatants were removed and analyzed by Luminex. Infections were carried out in triplicate. The solid line indicates the limit of detection for the assay. The broken line indicates the upper limit of detection.

Figure S8: Detection of IL-10 in Culture Supernatants of WT BMDM

Figure S9: Detection of IL-12p40/70 in Culture Supernatants of WT BMDM

Figure S10: Detection of IL-1α in Culture Supernatants of TLR4^-/- BMDM

C57Bl/6 TLR4^-/- BMDM were infected with the indicated MOI of either WT or mutant Salmonella. For arabinose positive conditions, both BMDM and Salmonella were grown in 0.2% arabinose for the entire experiment, including overnight and subculture of Salmonella. 6 hours after infection the supernatants were removed and analyzed by Luminex. Infections were carried out in triplicate. The solid line indicates the limit of detection for the assay. Mutants that induced no detectable cytokine are not included.

Figure S11: Detection of IL-1β in Culture Supernatants of TLR4^-/- BMDM

Figure S12: Detection of IL-4 in Culture Supernatants of TLR4^-/- BMDM

C57Bl/6 TLR4^-/- BMDM were infected with the indicated MOI of either WT or mutant Salmonella. For arabinose positive conditions, both BMDM and Salmonella were grown in 0.2% arabinose for the entire experiment, including overnight and subculture of Salmonella. 6 hours after infection the supernatants were removed and analyzed by Luminex. Infections were carried out in triplicate. The solid line indicates the limit of detection for the assay. Mutants that induced no detectable cytokine are not included.

Figure S13: Detection of IL-13 in Culture Supernatants of TLR4^-/- BMDM

Figure S14: Detection of KC in Culture Supernatants of TLR4^-/- BMDM

Figure S15: Detection of VEGF in Culture Supernatants of TLR4^-/- BMDM

Figure S16: Detection of MIP-1α in Culture Supernatants of TLR4^-/- BMDM

Figure S17: Detection of IL-10 in Culture Supernatants of TLR4^-/- BMDM

C57Bl/6 TLR-4^-/- BMDM were infected with the indicated MOI of either WT or mutant Salmonella. For arabinose positive conditions, both BMDM and Salmonella were grown in 0.2% arabinose for the entire experiment, including overnight and subculture of Salmonella. 6 hours after infection the supernatants were removed and analyzed by Luminex. Infections were carried out in triplicate. The solid line indicates the limit of detection for the assay.

Figure S18: Detection of IL-12p40/70 in Culture Supernatants of TLR4^-/- BMDM

NIHMS736425-supplement-supplement_1.pdf^{(1MB, pdf)}

Highlights.

Nine attenuated Salmonella mutants were assayed for cellular invasion and host cytokine production.
Every mutant examined gave a distinct cytokine fingerprint compared to WT infection.
IL-10 restricted growth of WT Salmonella in Bone Marrow Derived Macrophages.

Footnotes

We are delighted to provide this contribution to the Ray D. Owen issue of Cellular Immunology. We have chosen this contribution since it uses one of Owen's favorite scientific strategies, the use of genetics to probe important immunological questions. Here we combine host genetics with the genetics of Salmonella to ask how bacteria and hosts interact and how loss of function mutants in bacteria can result in novel cytokine responses from the host.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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