Flotillin-2 dampens T cell antigen-sensitivity and functionality.

T cell receptor (TCR) engagement triggers T cell responses, yet how TCR-mediated activation is regulated at the plasma membrane remains unclear. Here, we report that deleting the membrane scaffolding protein Flotillin-2 (Flot2) increases T cell antigen sensitivity, resulting in enhanced TCR signaling and effector function to weak TCR stimulation. T cell-specific Flot2-deficient mice exhibited reduced tumor growth and enhanced immunity to infection. Flot2-null CD4 + T cells exhibited increased T helper 1 polarization, proliferation, Nur77 induction, and phosphorylation of ZAP70 and LCK upon weak TCR stimulation, indicating a sensitized TCR-triggering threshold. Single cell-RNA sequencing suggested that Flot2 - null CD4 + T cells follow a similar route of activation as wild-type CD4 + T cells but exhibit higher occupancy of a discrete activation state under weak TCR stimulation. Given prior reports that TCR clustering influences sensitivity of T cells to stimuli, we evaluated TCR distribution with super-resolution microscopy. Flot2 ablation increased the number of surface TCR nanoclusters on naïve CD4 + T cells. Collectively, we posit that Flot2 modulates T cell functionality to weak TCR stimulation, at least in part, by regulating surface TCR clustering. Our findings have implications for improving T cell reactivity in diseases with poor antigenicity, such as cancer and chronic infections.

Oxidized Low-Density Lipoprotein Accumulation Suppresses Glycolysis and Attenuates the Macrophage Inflammatory Response by Diverting Transcription from the HIF-1α to the Nrf2 Pathway.

Lipid accumulation in macrophages (Mφs) is a hallmark of atherosclerosis, yet how lipid accumulation affects inflammatory responses through rewiring of Mφ metabolism is poorly understood. We modeled lipid accumulation in cultured wild-type mouse thioglycolate-elicited peritoneal Mφs and bone marrow-derived Mφs with conditional (Lyz2-Cre) or complete genetic deficiency of Vhl, Hif1a, Nos2, and Nfe2l2. Transfection studies employed RAW264.7 cells. Mφs were cultured for 24 h with oxidized low-density lipoprotein (oxLDL) or cholesterol and then were stimulated with LPS. Transcriptomics revealed that oxLDL accumulation in Mφs downregulated inflammatory, hypoxia, and cholesterol metabolism pathways, whereas the antioxidant pathway, fatty acid oxidation, and ABC family proteins were upregulated. Metabolomics and extracellular metabolic flux assays showed that oxLDL accumulation suppressed LPS-induced glycolysis. Intracellular lipid accumulation in Mφs impaired LPS-induced inflammation by reducing both hypoxia-inducible factor 1-α (HIF-1α) stability and transactivation capacity; thus, the phenotype was not rescued in Vhl-/- Mφs. Intracellular lipid accumulation in Mφs also enhanced LPS-induced NF erythroid 2-related factor 2 (Nrf2)-mediated antioxidative defense that destabilizes HIF-1α, and Nrf2-deficient Mφs resisted the inhibitory effects of lipid accumulation on glycolysis and inflammatory gene expression. Furthermore, oxLDL shifted NADPH consumption from HIF-1α- to Nrf2-regulated apoenzymes. Thus, we postulate that repurposing NADPH consumption from HIF-1α to Nrf2 transcriptional pathways is critical in modulating inflammatory responses in Mφs with accumulated intracellular lipid. The relevance of our in vitro models was established by comparative transcriptomic analyses, which revealed that Mφs cultured with oxLDL and stimulated with LPS shared similar inflammatory and metabolic profiles with foamy Mφs derived from the atherosclerotic mouse and human aorta.

DNASE1L3 enhances antitumor immunity and suppresses tumor progression in colon cancer.

DNASE1L3, an enzyme highly expressed in DCs, is functionally important for regulating autoimmune responses to self-DNA and chromatin. Deficiency of DNASE1L3 leads to development of autoimmune diseases in both humans and mice. However, despite the well-established causal relationship between DNASE1L3 and immunity, little is known about the involvement of DNASE1L3 in regulation of antitumor immunity, the foundation of modern antitumor immunotherapy. In this study, we identify DNASE1L3 as a potentially new regulator of antitumor immunity and a tumor suppressor in colon cancer. In humans, DNASE1L3 is downregulated in tumor-infiltrating DCs, and this downregulation is associated with poor patient prognosis and reduced tumor immune cell infiltration in many cancer types. In mice, Dnase1l3 deficiency in the tumor microenvironment enhances tumor formation and growth in several colon cancer models. Notably, the increased tumor formation and growth in Dnase1l3-deficient mice are associated with impaired antitumor immunity, as evidenced by a substantial reduction of cytotoxic T cells and a unique subset of DCs. Consistently, Dnase1l3-deficient DCs directly modulate cytotoxic T cells in vitro. To our knowledge, our study unveils a previously unknown link between DNASE1L3 and antitumor immunity and further suggests that restoration of DNASE1L3 activity may represent a potential therapeutic approach for anticancer therapy.

Meta-Analysis of COVID-19 BAL Single-Cell RNA Sequencing Reveals Alveolar Epithelial Transitions and Unique Alveolar Epithelial Cell Fates.

Single-cell RNA sequencing (scRNA-seq) of BAL cells has provided insights into coronavirus disease (COVID-19). However, reports have been limited by small patient cohorts. We performed a meta-analysis of BAL scRNA-seq data from healthy control subjects (n = 13) and patients with COVID-19 (n = 20), sourced from six independent studies (167,280 high-quality cells in total). Consistent with the source reports, increases in infiltrating leukocyte subtypes were noted, several with type I IFN signatures and unique gene expression signatures associated with transcellular chemokine signaling. Noting dramatic reductions of inferred NKX2-1 and NR4A1 activity in alveolar epithelial type II (AT-II) cells, we modeled pseudotemporal AT-II-to-AT-I progression. This revealed changes in inferred AT-II cell metabolic activity, increased transitional cells, and a previously undescribed AT-I state. This cell state was conspicuously marked by the induction of genes of the epidermal differentiation complex, including the cornified envelope protein SPRR3 (small proline-rich protein 3), upregulation of multiple KRT (keratin) genes, inferred mitochondrial dysfunction, and cell death signatures including apoptosis and ferroptosis. Immunohistochemistry of lungs from patients with COVID-19 confirmed upregulation and colocalization of KRT13 and SPRR3 in the distal airspaces. Forced overexpression of SPRR3 in human alveolar epithelial cells ex vivo did not activate caspase-3 or upregulate KRT13, suggesting that SPRR3 marks an AT-I cornification program in COVID-19 but is not sufficient for phenotypic changes.

An in vitro model of human hematopoiesis identifies a regulatory role for the aryl hydrocarbon receptor.

In vitro models to study simultaneous development of different human immune cells and hematopoietic lineages are lacking. We identified and characterized, using single-cell methods, an in vitro stromal cell-free culture system of human hematopoietic stem and progenitor cell (HSPC) differentiation that allows concurrent development of multiple immune cell lineages. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor influencing many biological processes in diverse cell types. Using this in vitro model, we found that AHR activation by the highly specific AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin, drives differentiation of human umbilical cord blood-derived CD34+ HSPCs toward monocytes and granulocytes with a significant decrease in lymphoid and megakaryocyte lineage specification that may lead to reduced immune competence. To our knowledge, we also discovered for the first time, using single-cell modalities, that AHR activation decreased the expression of BCL11A and IRF8 in progenitor cells, which are critical genes involved in hematopoietic lineage specification processes at both transcriptomic and protein levels. Our in vitro model of hematopoiesis, coupled with single-cell tools, therefore allows for a better understanding of the role played by AHR in modulating hematopoietic differentiation.

25-Hydroxycholesterol exacerbates vascular leak during acute lung injury.

Cholesterol-25-hydroxylase (CH25H), the biosynthetic enzyme for 25-hydroxycholesterol (25HC), is most highly expressed in the lung, but its role in lung biology is poorly defined. Recently, we reported that Ch25h is induced in monocyte-derived macrophages recruited to the airspace during resolution of lung inflammation and that 25HC promotes liver X receptor-dependent (LXR-dependent) clearance of apoptotic neutrophils by these cells. Ch25h and 25HC are, however, also robustly induced by lung-resident cells during the early hours of lung inflammation, suggesting additional cellular sources and targets. Here, using Ch25h-/- mice and exogenous 25HC in lung injury models, we provide evidence that 25HC sustains proinflammatory cytokines in the airspace and augments lung injury, at least in part, by inducing LXR-independent endoplasmic reticulum stress and endothelial leak. Suggesting an autocrine effect in endothelium, inhaled LPS upregulates pulmonary endothelial Ch25h, and non-hematopoietic Ch25h deletion is sufficient to confer lung protection. In patients with acute respiratory distress syndrome, airspace 25HC and alveolar macrophage CH25H were associated with markers of microvascular leak, endothelial activation, endoplasmic reticulum stress, inflammation, and clinical severity. Taken together, our findings suggest that 25HC deriving from and acting on different cell types in the lung communicates distinct, temporal LXR-independent and -dependent signals to regulate inflammatory homeostasis.

IRGM1 links mitochondrial quality control to autoimmunity.

Mitochondrial abnormalities have been noted in lupus, but the causes and consequences remain obscure. Autophagy-related genes ATG5, ATG7 and IRGM have been previously implicated in autoimmune disease. We reasoned that failure to clear defective mitochondria via mitophagy might be a foundational driver in autoimmunity by licensing mitochondrial DNA-dependent induction of type I interferon. Here, we show that mice lacking the GTPase IRGM1 (IRGM homolog) exhibited a type I interferonopathy with autoimmune features. Irgm1 deletion impaired the execution of mitophagy with cell-specific consequences. In fibroblasts, mitochondrial DNA soiling of the cytosol induced cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-dependent type I interferon, whereas in macrophages, lysosomal Toll-like receptor 7 was activated. In vivo, Irgm1-/- tissues exhibited mosaic dependency upon nucleic acid receptors. Whereas salivary and lacrimal gland autoimmune pathology was abolished and lung pathology was attenuated by cGAS and STING deletion, pancreatic pathology remained unchanged. These findings reveal fundamental connections between mitochondrial quality control and tissue-selective autoimmune disease.

Metabolic heterogeneity underlies reciprocal fates of TH17 cell stemness and plasticity.

A defining feature of adaptive immunity is the development of long-lived memory T cells to curtail infection. Recent studies have identified a unique stem-like T-cell subset amongst exhausted CD8-positive T cells in chronic infection1-3, but it remains unclear whether CD4-positive T-cell subsets with similar features exist in chronic inflammatory conditions. Amongst helper T cells, TH17 cells have prominent roles in autoimmunity and tissue inflammation and are characterized by inherent plasticity4-7, although how such plasticity is regulated is poorly understood. Here we demonstrate that TH17 cells in a mouse model of autoimmune disease are functionally and metabolically heterogeneous; they contain a subset with stemness-associated features but lower anabolic metabolism, and a reciprocal subset with higher metabolic activity that supports transdifferentiation into TH1-like cells. These two TH17-cell subsets are defined by selective expression of the transcription factors TCF-1 and T-bet, and by discrete levels of CD27 expression. We also identify signalling via the kinase complex mTORC1 as a central regulator of TH17-cell fate decisions by coordinating metabolic and transcriptional programmes. TH17 cells with disrupted mTORC1 signalling or anabolic metabolism fail to induce autoimmune neuroinflammation or to develop into TH1-like cells, but instead upregulate TCF-1 expression and acquire stemness-associated features. Single-cell RNA sequencing and experimental validation reveal heterogeneity in fate-mapped TH17 cells, and a developmental arrest in the TH1 transdifferentiation trajectory upon loss of mTORC1 activity or metabolic perturbation. Our results establish that the dichotomy of stemness and effector function underlies the heterogeneous TH17 responses and autoimmune pathogenesis, and point to previously unappreciated metabolic control of plasticity in helper T cells.

Hippo/Mst signalling couples metabolic state and immune function of CD8α+ dendritic cells.

Dendritic cells orchestrate the crosstalk between innate and adaptive immunity. CD8α+ dendritic cells present antigens to CD8+ T cells and elicit cytotoxic T cell responses to viruses, bacteria and tumours 1 . Although lineage-specific transcriptional regulators of CD8α+ dendritic cell development have been identified 2 , the molecular pathways that selectively orchestrate CD8α+ dendritic cell function remain elusive. Moreover, metabolic reprogramming is important for dendritic cell development and activation3,4, but metabolic dependence and regulation of dendritic cell subsets are largely uncharacterized. Here we use a data-driven systems biology algorithm (NetBID) to identify a role of the Hippo pathway kinases Mst1 and Mst2 (Mst1/2) in selectively programming CD8α+ dendritic cell function and metabolism. Our NetBID analysis reveals a marked enrichment of the activities of Hippo pathway kinases in CD8α+ dendritic cells relative to CD8α- dendritic cells. Dendritic cell-specific deletion of Mst1/2-but not Lats1 and Lats2 (Lats1/2) or Yap and Taz (Yap/Taz), which mediate canonical Hippo signalling-disrupts homeostasis and function of CD8+ T cells and anti-tumour immunity. Mst1/2-deficient CD8α+ dendritic cells are impaired in presentation of extracellular proteins and cognate peptides to prime CD8+ T cells, while CD8α- dendritic cells that lack Mst1/2 have largely normal function. Mechanistically, compared to CD8α- dendritic cells, CD8α+ dendritic cells exhibit much stronger oxidative metabolism and critically depend on Mst1/2 signalling to maintain bioenergetic activities and mitochondrial dynamics for their functional capacities. Further, selective expression of IL-12 by CD8α+ dendritic cells depends on Mst1/2 and the crosstalk with non-canonical NF-κB signalling. Our findings identify Mst1/2 as selective drivers of CD8α+ dendritic cell function by integrating metabolic activity and cytokine signalling, and highlight that the interplay between immune signalling and metabolic reprogramming underlies the unique functions of dendritic cell subsets.

Critical roles of mTORC1 signaling and metabolic reprogramming for M-CSF-mediated myelopoiesis.

Myelopoiesis is necessary for the generation of mature myeloid cells during homeostatic turnover and immunological insults; however, the metabolic requirements for this process remain poorly defined. Here, we demonstrate that myelopoiesis, including monocyte and macrophage differentiation, requires mechanistic target of rapamycin complex 1 (mTORC1) signaling and anabolic metabolism. Loss of mTORC1 impaired myelopoiesis under steady state and dampened innate immune responses against Listeria monocytogenes infection. Stimulation of hematopoietic progenitors with macrophage colony-stimulating factor (M-CSF) resulted in mTORC1-dependent anabolic metabolism, which in turn promoted expression of M-CSF receptor and transcription factors PU.1 and IRF8, thereby constituting a feed-forward loop for myelopoiesis. Mechanistically, mTORC1 engaged glucose metabolism and initiated a transcriptional program involving Myc activation and sterol biosynthesis after M-CSF stimulation. Perturbation of glucose metabolism or disruption of Myc function or sterol biosynthesis impaired myeloid differentiation. Integrative metabolomic and genomic profiling further identified one-carbon metabolism as a central node in mTORC1-dependent myelopoiesis. Therefore, the interplay between mTORC1 signaling and metabolic reprogramming underlies M-CSF-induced myelopoiesis.

Contributions of nonhematopoietic cells and mediators to immune responses: implications for immunotoxicology.

Immunotoxicology assessments have historically focused on the effects that xenobiotics exhibit directly on immune cells. These studies are invaluable as they identify immune cell targets and help characterize mechanisms and/or adverse outcome pathways of xenobiotics within the immune system. However, leukocytes can receive environmental cues by cell-cell contact or via released mediators from cells of organs outside of the immune system. These organs include, but are not limited to, the mucosal areas such as the lung and the gut, the liver, and the central nervous system. Homeostatic perturbation in these organs induced directly by toxicants can initiate and alter the outcome of local and systemic immunity. This review will highlight some of the identified nonimmune influences on immune homeostasis and provide summaries of how immunotoxic mechanisms of selected xenobiotics involve nonimmune cells or mediators. Thus, this review will identify data gaps and provide possible alternative mechanisms by which xenobiotics alter immune function that could be considered during immunotoxicology safety assessment.

Tsc1 promotes the differentiation of memory CD8+ T cells via orchestrating the transcriptional and metabolic programs.

Memory CD8(+) T cells are an essential component of protective immunity. Signaling via mechanistic target of rapamycin (mTOR) has been implicated in the regulation of the differentiation of effector and memory T cells. However, little is understood about the mechanisms that control mTOR activity, or the effector pathways regulated by mTOR. We describe here that tuberous sclerosis 1 (Tsc1), a regulator of mTOR signaling, plays a crucial role in promoting the differentiation and function of memory CD8(+) T cells in response to Listeria monocytogenes infection. Mice with specific deletion of Tsc1 in antigen-experienced CD8(+) T cells evoked normal effector responses, but were markedly impaired in the generation of memory T cells and their recall responses to antigen reexposure in a cell-intrinsic manner. Tsc1 deficiency suppressed the generation of memory-precursor effector cells while promoting short-lived effector cell differentiation. Transcriptome analysis indicated that Tsc1 coordinated gene expression programs underlying immune function, transcriptional regulation, and cell metabolism. Furthermore, Tsc1 deletion led to excessive mTORC1 activity and dysregulated glycolytic and oxidative metabolism in response to IL-15 stimulation. These findings establish a Tsc1-mediated checkpoint in linking immune signaling and cell metabolism to orchestrate memory CD8(+) T-cell development and function.

Cannabidiol (CBD) enhances lipopolysaccharide (LPS)-induced pulmonary inflammation in C57BL/6 mice.

Cannabidiol (CBD) is a plant-derived cannabinoid that has been predominantly characterized as anti-inflammatory. However, it is clear that immune effects of cannabinoids can vary with cannabinoid concentration, or type or magnitude of immune stimulus. The present studies demonstrate that oral administration of CBD enhanced lipopolysaccharide (LPS)-induced pulmonary inflammation in C57BL/6 mice. The enhanced inflammatory cell infiltrate as observed in bronchoalveolar lavage fluid (BALF) was comprised mainly of neutrophils, with some monocytes. Concomitantly, CBD enhanced pro-inflammatory cytokine mRNA production, including tumor necrosis factor-α (Tnfa), interleukins (IL)-5 and -23 (Il6, Il23), and granulocyte colony stimulating factor (Gcsf). These results demonstrate that the CBD-mediated enhancement of LPS-induced pulmonary inflammation is mediated at the level of transcription of a variety of pro-inflammatory genes. The significance of these studies is that CBD is part of a therapeutic currently in use for spasticity and pain in multiple sclerosis patients, and therefore it is important to further understand mechanisms by which CBD alters immune function.

The effects of targeted deletion of cannabinoid receptors CB1 and CB2 on intranasal sensitization and challenge with adjuvant-free ovalbumin.

The mechanisms by which cannabinoid receptors CB(1) and CB(2) modulate immune function are not fully elucidated. Critical tools for the determination of the role of both receptors in the immune system are CB(1)/CB(2) double null mice (CB(1)/CB(2) null), and previous studies have shown that CB(1)/CB(2) null mice exhibit exaggerated responses to various immunological stimuli. The objective of these studies was to determine the magnitude to which CB(1)/CB(2) null mice responded to the respiratory allergen ovalbumin (OVA) as compared with wild-type C57BL/6 mice. The authors determined that in the absence of adjuvant, both wild-type and CB(1)/CB(2) null mice mounted a marked response to intranasally instilled OVA as assessed by inflammatory cell infiltrate in the bronchoalveolar lavage fluid (BALF), eosinophilia, induction of mucous cell metaplasia, and IgE production. Many of the endpoints measured in response to OVA were similar in wild-type versus CB(1)/CB(2) null mice, with exceptions being modest reductions in OVA-induced IgE and attenuation of BALF neutrophilia in CB(1)/CB(2) null mice as compared with wild-type mice. These results suggest that T-cell responses are not universally exaggerated in CB(1)/CB(2) null mice.

Effects of targeted deletion of cannabinoid receptors CB1 and CB2 on immune competence and sensitivity to immune modulation by Delta9-tetrahydrocannabinol.

The role of cannabinoid receptors, CB1 and CB2, in immune competence and modulation by Delta9-tetrahydrocannabinol (Delta9-THC) was investigated in CB1(-/-)/CB2(-/-) mice. Immunofluorescence analysis of splenic leukocytes showed no significant differences in the percentage of T cell subsets, B cells, or macrophages between wild-type and CB1(-/-)/CB2(-/-) mice. Lymphoproliferative control responses to PHA, phorbol ester plus ionomycin, or LPS and sensitivity to suppression by Delta9-THC showed no profound differences between the two genotypes, although some differences were observed in control baseline responses. Likewise, similar control responses and sensitivity to Delta9-THC were observed in mixed lymphocyte responses (MLR) and in IL-2 and IFN-gamma production in both genotypes. Conversely, humoral immune responses showed a markedly different profile of activity. Delta9-THC suppressed the in vivo T cell-dependent, anti-sheep RBC (anti-sRBC) IgM antibody-forming cell (AFC) response in wild-type but not in CB1(-/-)/CB2(-/-) mice, and the in vitro anti-sRBC IgM response in CB1(-/-)/CB2(-/-) splenocytes was too low to rigorously assess CB1/CB2 involvement in modulation by Delta9-THC. Conversely, comparable in vitro IgM AFC control responses to LPS and CD40 ligand (CD40L) activation were observed in the two genotypes. Interestingly, LPS-induced IgM responses were refractory to suppression by Delta9-THC, regardless of genotype, and CD40L-induced IgM responses were only suppressed by Delta9-THC in wild-type but not in CB1(-/-)/CB2(-/-) B cells. Collectively, we demonstrate differential involvement of CB1 and/or CB2 in immune modulation by Delta9-THC and in some control responses. Moreover, CB1/CB2 involvement was observed in humoral responses requiring CD40-initiated signaling for suppression by Delta9-THC.

Targeted deletion of cannabinoid receptors CB1 and CB2 produced enhanced inflammatory responses to influenza A/PR/8/34 in the absence and presence of Delta9-tetrahydrocannabinol.

We have previously reported that Delta-9-tetrahydrocannabinol (Delta(9)-THC)-treated mice challenged with influenza virus A/PR/8/34 (PR8) developed increased viral hemagglutinin 1 (H1) mRNA levels and decreased monocyte and lymphocyte recruitment to the pulmonary airways when compared with mice challenged with PR8 alone. The objective of the present study was to examine the role of cannabinoid (CB(1)/CB(2)) receptors in mediating the effects of Delta(9)-THC on immune and epithelial cell responses to PR8. In the current study, Delta(9)-THC-treated CB(1)/CB(2) receptor null (CB(1)-/-/CB(2)-/-) and wild-type mice infected with PR8 had marked increases in viral H1 mRNA when compared with CB(1)-/-/CB(2)-/- and wild-type mice challenged with PR8 alone. However, the magnitude of the H1 mRNA levels was greatly reduced in CB(1)-/-/CB(2)-/- mice as compared with wild-type mice. In addition, Delta(9)-THC-treated CB(1)-/-/CB(2)-/- mice infected with PR8 had increased CD4+ T cells and IFN-gamma in bronchoalveolar lavage fluid with greater pulmonary inflammation when compared with Delta(9)-THC-treated wild-type mice infected with PR8. Delta(9)-THC treatment of CB(1)-/-/CB(2)-/- mice in the presence or absence of PR8 challenge also developed greater amounts of mucous cell metaplasia in the affected bronchiolar epithelium. Collectively, the immune and airway epithelial cell responses to PR8 challenge in Delta(9)-THC-treated CB(1)-/-/CB(2)-/- and wild-type mice indicated the involvement of CB(1)/CB(2) receptor-dependent and -independent mechanisms.

Modulation of airway responses to influenza A/PR/8/34 by Delta9-tetrahydrocannabinol in C57BL/6 mice.

Delta(9)-tetrahydrocannabinol (Delta(9)-THC) has been widely established as a modulator of host immune responses. Accordingly, the objective of the present study was to examine the effects of Delta(9)-THC on the immune response within the lungs and associated changes in the morphology of the bronchiolar epithelium after one challenge with a nonlethal dose of the influenza virus A/PR/8 (PR8). C57BL/6 mice were treated by oral gavage with Delta(9)-THC and/or vehicle (corn oil) for 5 consecutive days. On day 3, mice were instilled intranasally with 50 plaque-forming units of PR8 and/or vehicle (saline) 4 h before Delta(9)-THC exposure. Mice were subsequently killed 7 and 10 days postinfection (dpi). Viral hemagglutinin 1 (H1) mRNA levels in the lungs were increased in a dose-dependent manner with Delta(9)-THC treatment. Enumeration of inflammatory cell types in bronchoalveolar lavage fluid showed an attenuation of macrophages and CD4(+) and CD8(+) T cells in Delta(9)-THC-treated mice compared with controls. Likewise, the magnitude of inflammation and virus-induced mucous cell metaplasia, as assessed by histopathology, was reduced in Delta(9)-THC-treated mice by 10 dpi. Collectively, these results suggest that Delta(9)-THC treatment increased viral load, as assessed by H1 mRNA levels, through a decrease in recruitment of macrophages and lymphocytes, particularly CD4(+) and CD8(+) T cells, to the lung.