Background & Aims: Liver macrophages are heterogeneous and play an important role in alcohol-associated liver disease (ALD) but there is limited understanding of the functions of specific macrophage subsets in the disease. We used a Western Diet Alcohol (WDA) mouse model of ALD to examine the hepatic myeloid cell compartment by scRNA seq and targeted Kupffer cell (KC) ablation to understand the diversity and function of liver macrophages in ALD. Approach and Results: In the WDA liver, KCs and infiltrating monocytes/macrophages (IMs) each represented about 50% of the myeloid pool. Five major KC clusters all expressed genes associated with receptor mediated endocytosis and lipid metabolism, but most were predicted to be non-inflammatory and antifibrotic with one minor KC cluster having a pro-inflammatory and extracellular matrix degradation gene signature. IM clusters, in contrast, were predicted to be pro-inflammatory and pro-fibrotic. In vivo diphtheria toxin based selective KC ablation during alcohol exposure resulted in a liver failure phenotype with increases in PT/INR and bilirubin, loss of differentiated hepatocyte gene expression, and an increase in expression of hepatocyte progenitor markers such as EpCAM, CK-7 and Igf2bp3. Gene set enrichment analysis of whole liver RNAseq from the KC ablated WDA mice showed a similar pattern as seen in human alcoholic hepatitis. Conclusions: In this ALD model, KCs are anti-inflammatory and are critical for maintenance of hepatocyte differentiation. IMs are largely pro-inflammatory and contribute more to liver fibrosis. Future targeting of specific macrophage subsets may provide new approaches to treatment of liver failure and fibrosis in ALD. Export

Clinical trials for reducing fibrosis in steatotic liver disease (SLD) have targeted macrophages with variable results. We evaluated intrahepatic macrophages in patients with SLD to determine if activity scores or fibrosis stages influenced phenotypes and expression of druggable targets, such as CCR2 and galectin-3. Liver biopsies from controls or patients with minimal or advanced fibrosis were subject to gene expression analysis using nCounter to determine differences in macrophage-related genes (n = 30). To investigate variability among individual patients, we compared additional biopsies by staining them with multiplex antibody panels (CD68/CD14/CD16/CD163/Mac387 or CD163/CCR2/galectin-3/Mac387) followed by spectral imaging and spatial analysis. Algorithms that utilize deep learning/artificial intelligence were applied to create cell cluster plots, phenotype profile maps, and to determine levels of protein expression (n = 34). Several genes known to be pro-fibrotic (e.g. CD206, TREM2, CD163, and ARG1) showed either no significant differences or significantly decreased with advanced fibrosis. Although marked variability in gene expression was observed in individual patients with cirrhosis, several druggable targets and their ligands (e.g. CCR2, CCR5, CCL2, CCL5, and LGALS3) were significantly increased when compared to patients with minimal fibrosis. Antibody panels identified populations that were significantly increased (e.g. Mac387+), decreased (e.g. CD14+), or enriched (e.g. interactions of Mac387) in patients that had progression of disease or advanced fibrosis. Despite heterogeneity in patients with SLD, several macrophage phenotypes and druggable targets showed a positive correlation with increasing NAFLD activity scores and fibrosis stages. Patients with SLD have markedly varied macrophage- and druggable target-related gene and protein expression in their livers. Several patients had relatively high expression, while others were like controls. Overall, patients with more advanced disease had significantly higher expression of CCR2 and galectin-3 at both the gene and protein levels. Appreciating individual differences within the hepatic microenvironment of patients with SLD may be paramount to developing effective treatments. These results may explain why such a small percentage of patients have responded to macrophage-targeting therapies and provide additional support for precision medicine-guided treatment of chronic liver diseases.

Tick-borne rickettsial pathogens (TBRP) are important causes of infections in both dogs and humans. Dogs play an important role as a biological host for several tick species and can serve as sentinels for rickettsial infections. Our aim was to determine the presence of TBRP in dogs and in dog-associated ticks and their potential risk to human diseases in Medellin, Colombia. DNA for E. canis (16S rRNA and dsb) and A. platys (groEl) was detected in 17.6% (53/300) and 2.6% (8/300) of dogs, respectively. Antibodies against Ehrlichia spp. 82 (27.3%) and Anaplasma spp. 8 (2.6%) were detected in dogs. Antibody reactivity against both agents were found in 16 dogs (5.3%). Eight dogs showed antibody for Rickettsia spp. with titers that suggest 3 of them had a probable exposure to R. parkeri. Rhipicephalus sanguineus s.l. (178/193) was the main tick in dogs, followed by R. microplus (15/193). The minimum infection rates (MIR) in R. sanguineus were 11.8% for E. canis and 3.4% for A. platys. E. canis and A. platys are the main TBRP infecting dogs and ticks and R. sanguineus s.l. is likely involved in the transmission of both agents. Interestingly, we found serological evidence of exposure in dogs for spotted fever group rickettsiae.

Although rickettsiae can cause life-threatening infections in humans worldwide, no licensed vaccine is currently available. To evaluate the suitability of live-attenuated vaccine candidates against rickettsioses, we generated a Rickettsia parkeri mutant RPATATE_0245::pLoxHimar (named 3A2) by insertion of a modified pLoxHimar transposon into the gene encoding a phage integrase protein. For visualization and selection, R. parkeri 3A2 expressed mCherry fluorescence and resistance to spectinomycin. Compared to the parent wild type (WT) R. parkeri, the virulence of R. parkeri 3A2 was significantly attenuated as demonstrated by significantly smaller size of plaque, failure to grow in human macrophage-like cells, rapid elimination of Rickettsia and ameliorated histopathological changes in tissues in intravenously infected mice. A single dose intradermal (i.d.) immunization of R. parkeri 3A2 conferred complete protection against both fatal R. parkeri and R. conorii rickettsioses in mice, in association with a robust and durable rickettsiae-specific IgG antibody response. In summary, the disruption of RPATATE_0245 in R. parkeri resulted in a mutant with a significantly attenuated phenotype, potent immunogenicity and protective efficacy against two spotted fever group rickettsioses. Overall, this proof-of-concept study highlights the potential of R. parkeri mutants as a live-attenuated and multivalent vaccine platform in response to emergence of life-threatening spotted fever rickettsioses.

Autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) are immune-mediated liver diseases (IMLDs) that are diagnosed by a combination of clinical, serologic, and histologic features. Diagnosis may be challenging, particularly when patients have mixed features of both AIH and PBC, a disease often called overlap syndrome (OS). In addition, many patients have refractory disease. We hypothesized that adding molecular testing to the current diagnostic criteria would provide an additional tool that could assist in correctly classifying patients. RNA was isolated from liver biopsies from patients with AIH (n = 16), PBC (n = 13), OS (n = 8), drug-induced/serology-negative AIH (AIH DI/Ser-neg, n = 6), or controls (n = 10). Gene expression was determined using an nCounter Sprint Profiler, and principal component analysis delineated distinct clusters for patients with an inflammatory profile due to AIH, PBC, and AIH DI/Ser-neg. Two patients with minimal histologic features of PBC clustered with the control group, and 2 patients with predominantly AIH and minimal PBC features clustered with the PBC group. A patient with OS who received treatment for both conditions showed no disease progression, whereas a patient with OS treated solely for AIH failed to respond. Conversely, one of the gene signatures from a patient diagnosed with PBC fell within the AIH group. This patient did not respond to treatment with ursodiol and ultimately required liver replacement. These findings suggest that the IMLD initially diagnosed in these patients may have been incorrectly classified. As expected, molecular analysis could not identify a distinct cluster for patients diagnosed with OS, and these had variable gene signatures that fell throughout the identified AIH or PBC groups. Cluster analysis was also able to distinguish patients with disease progression from non-progressors with mild disease who responded to treatment. In summary, gene expression analysis may assist in confirming the type of IMLD, especially when the diagnosis is unclear. Combining molecular testing with existing criteria could provide an additional diagnostic tool, improving patient care and response to treatment.